EP1411761B1 - Transporteurs de metabolites - Google Patents

Transporteurs de metabolites Download PDF

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EP1411761B1
EP1411761B1 EP02769376A EP02769376A EP1411761B1 EP 1411761 B1 EP1411761 B1 EP 1411761B1 EP 02769376 A EP02769376 A EP 02769376A EP 02769376 A EP02769376 A EP 02769376A EP 1411761 B1 EP1411761 B1 EP 1411761B1
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promoter
plant
nucleic acid
gene
protein
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EP1411761A2 (fr
EP1411761A4 (fr
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Henry E. Valentin
Toni Voelker
Wei Zheng
Thomas J. Savage
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Monsanto Technology LLC
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Monsanto Technology LLC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine

Definitions

  • the present invention is in the field of plant genetics and biochemistry. More specifically, the invention relates to genes associated with nucleotide triphosphate transport.
  • the present invention provides and includes nucleic acid molecules, proteins, and antibodies associated with the genes involved in nucleotide triphosphate transport.
  • the present invention also includes transgenic plants modified to express proteins associated with nucleotide triphosphate transport.
  • DNA molecules are provided that constitute fragments of the genome of a plant, and polypeptides encoded thereby.
  • the DNA molecules are useful for specifying a gene product in cells, either as a promoter or as a protein coding sequence or as an UTR or as a 3'termination sequence, and are also useful in controlling the behavior of a gene in the chromosome, in controlling the expression of a gene or as tools for genetic mapping, recognizing or isolating identical or related DNA fragments, or identification of a particular individual organism, or for clustering of a group of organisms with a common trait.
  • Tocopherols are an essential component of mammalian diets. Epidemiological evidence indicates that tocopherol supplementation can result in decreased risk for cardiovascular disease and cancer, can aid in immune function, and is associated with prevention or retardation of a number of degenerative disease processes in humans ( Traber and Sies, Annu. Rev. Nutr. 16:321-347 (1996 )). Tocopherol functions, in part, by stabilizing the lipid bilayer of biological membranes ( Skrypin and Kagan, Biochim. Biophys. Acta 815:209 (1995 ); Kagan, N.Y Acad Sci. p 121, (1989 ); Gomez-Fernandez et al., Ann. N.Y. Acad. Sci.
  • ⁇ -Tocopherol often referred to as vitamin E, belongs to a class of lipid-soluble antioxidants that includes ⁇ , ⁇ , ⁇ , and ⁇ -tocopherols and ⁇ , ⁇ , ⁇ , and ⁇ -tocotrienols. Although ⁇ , ⁇ , ⁇ , and ⁇ -tocopherols and ⁇ , ⁇ , ⁇ , and ⁇ -tocotrienois are sometimes referred to collectively as "vitamin E," vitamin E is more appropriately defined chemically as ⁇ -tocopherol.
  • ⁇ -tocopherol is significant for human health, in part because it is readily absorbed and retained by the body, and has a higher degree of bioactivity than other tocopherol species ( Traber and Sies, Annu. Rev. Nutr. 16:321-347 (1996 )). However, other tocopherols such as ⁇ , ⁇ , and ⁇ -tocopherols, also have significant health and nutritional benefits.
  • Tocopherols are primarily synthesized only by plants and certain other photosynthetic organisms, including cyanobacteria. As a result, mammalian dietary tocopherols are obtained almost exclusively from these sources. Plant tissues vary considerably in total tocopherol content and tocopherol composition, with ⁇ -tocopherol the predominant tocopherol species found in green, photosynthetic plant tissues. Leaf tissue can contain from 10-50 ⁇ g of total tocopherols per gram fresh weight, but most of the world's major staple crops ( e.g.
  • the recommended daily dietary intake of 15-30 mg of vitamin E is quite difficult to achieve from the average American diet. For example, it would take over 750 grams of spinach leaves, in which ⁇ -tocopherol comprises 60% of total tocopherols, or 200-400 grams of soybean oil to satisfy this recommended daily vitamin E intake. While it is possible to augment the diet with supplements, most of these supplements contain primarily synthetic vitamin E, having six stereoisomers, whereas natural vitamin E is predominantly composed of only a single isomer. Furthermore, supplements tend to be relatively expensive, and the general population is disinclined to take vitamin supplements on a regular basis. Therefore, there is a need in the art for compositions and methods that either increase the total tocopherol production or increase the relative percentage of -tocopherol produced by plants.
  • tocpherols In addition to the health benefits of tocpherols, increased ⁇ -tocopherol levels in crops have been associated with enhanced stability and extended shelf life of plant products ( Peterson, Cereal-Chem 72(1):21-24 (1995 ); Ball, Fat-soluble vitamin assays in food analysis. A comprehensive review, London, Elsevier Science Publishers Ltd. (1985 )). Further, tocopherol supplementation of swine, beef, and poultry feeds has been shown to significantly increase meat quality and extend the shelf life of post-processed meat products by retarding post-processing lipid oxidation, which contributes to undesirable flavor components ( Sante and Lacourt, J. Sci. Food Agric. 65(4):503-507-(1994 ); Buckley et al., J. ofAnimal Science 73:3122-3130 (1995 )).
  • the plastids of higher plants exhibit interconnected biochemical pathways leading to secondary metabolites including tocopherols.
  • the tocopherol biosynthetic pathway in higher plants involves condensation of homogentisic acid and phytylpyrophosphate to form 2-methyl-6 phytylplastoquinol ( Fiedler et al., Planta 155: 511-515 (1982 ); Soll et al., Arch. Biochem. Biophys. 204: 544-550 (1980 ); Marshall et.al., Phytochem. 24: 1705-1711 (1985 )).
  • This plant tocopherol pathway can be divided into four parts: 1) synthesis of homogentistic acid, which contributes to the aromatic ring of tocopherol; 2) synthesis of phytylpyrophosphate, which contributes to the side chain of tocopherol; 3) cyclization, which plays a role in chirality and chromanol substructure of the vitamin E family; 4) and S-adenosyl methionine dependent methylation of an aromatic ring, which affects the relative abundance of each of the tocopherol species.
  • Homogentisic acid is the common precursor to both tocopherols and plastoquinones. In at least some bacteria the synthesis of homogentesic acid is believed to occur via the conversion of chorismate to prephenate and then to p-hydroxyphenylpyruvate via a bifunctional prephenate dehydrogenase.
  • bifunctional bacterial prephenate dehydrogenase enzymes include the proteins encoded by the TyrA genes of Erwinia herbicola and Escherichia coli. The TyrA gene product catalyzes the production of prephenate from chorismate, as well as the subsequent dehydrogenation of prephenate to form p-hydroxyphenylpyruvate (p-HPP), the immediate precursor to homogentistic acid.
  • p-HPP is then converted to homogentistic acid by hydroxyphenylpyruvate dioxygenase (HPPD).
  • HPPD hydroxyphenylpyruvate dioxygenase
  • plants are reported to lack prephenate dehydrogenase activity, and it is further reported that the synthesis of homogentesic acid from chorismate occurs via the synthesis and conversion of the intermediate arogenate. Since pathways involved in homogentesic acid synthesis are also responsible for tyrosine formation, any alterations in these pathways can also result in the alteration in tyrosine synthesis and the synthesis of other aromatic amino acids.
  • Tocopherols are a member of the class of compounds referred to as the isoprenoids.
  • Other isoprenoids include carotenoids, gibberellins, terpenes, chlorophyll and abscisic acid.
  • IPP isopentenyl diphosphate
  • Cytoplasmic and plastid-based pathways to generate IPP have been reported.
  • the cytoplasmic based pathway involves the enzymes acetoacetyl CoA thiolase, HMGCoA synthase, HMGCoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate pyrophosphate decarboxylase.
  • 1-Deoxyxylulose 5-phosphate can be further converted into 2- C- methylerythritol 4-phosphate ( Arigoni et al., Proc. Natl. Acad. Sci. USA, 94:10600-10605 (1997 )) by a reductoisomerase catalyzed by the dxr gene ( Bouvier et al., Plant Physiol, 117:1421-1431 (1998 ); and Rohdich et al., Proc. Natl. Acad. Sci. USA, 96:11755-11763 (1999 )).
  • Reported genes in the MEP pathway also include ygbP, which catalyzes the conversion of 2-C-methylerythritol 4-phosphate into its respective cytidyl pyrophosphate derivative and ygbB, which catalyzes the conversion of 4-phosphocytidyl-2C-methyl-D-erythritol into 2C-methyl-D-erythritol, 3, 4-cyclophosphate.
  • ygbP which catalyzes the conversion of 2-C-methylerythritol 4-phosphate into its respective cytidyl pyrophosphate derivative
  • ygbB which catalyzes the conversion of 4-phosphocytidyl-2C-methyl-D-erythritol into 2C-methyl-D-erythritol, 3, 4-cyclophosphate.
  • IPP is formed by the MEP pathway, it is converted to GGDP by GGDP synthase, and then to phytylpyrophosphate, which is the central constituent of the tocopherol side chain.
  • Homogentisic acid is combined with either phytyl-pyrophosphate or solanyl-pyrophosphate by phytyl/prenyl transferase forming 2-methyl-6-phytyl plastoquinol or 2-methyl-6-solanyl plastoquinol respectively.
  • 2-methyl-6-solanyl plastoquinol is a precursor to the biosynthesis of plastoquinones, while 2-methyl-6-phytyl plastoquinol is ultimately converted to tocopherol.
  • a major structural difference between each of the tocopherol subtypes is the position of the methyl groups around the phenyl ring.
  • 2-methyl-6-phytyl plastoquinol and 2-methyl-6-solanyl plastoquinol serve as substrates for 2-methyl-6-phytylplatoquinol/2-methyl-6-solanylplastoquinol-9 methyltransferase (Methyl Transferase 1; MT1), which catalyzes the formation of plastoquinol-9 and -tocopherol respectively, by methylation of the 7 position. Subsequent methylation at the 5 position of -tocopherol by -methyl-transferase generates the biologically active -tocopherol.
  • MT1 Metal Transferase 1
  • Adenylate transporters supply nucleotide triphosphates (including ATP and CTP) into the cell or cellular compartment for use in biosynthetic or metabolic processes.
  • nucleotide triphosphates including ATP and CTP
  • a putative adenylate transporter sequence from Arabidopsis thaliana has been reported in the literature ( Saint Guily, et al., Plant Physiol., 100(2):1069-1071 (1992 )).
  • nucleic acid molecules encoding enzymes involved in nucleotide triphosphate transport, as well as related enzymes and antibodies for the enhancement or alteration of tocopherol production in plants.
  • transgenic organisms expressing those nucleic acid molecules involved in nucleotide triphosphate transport, which are capable of nutritionally enhancing food and feed sources.
  • the present invention includes and provides a nucleic acid molecule that encodes an adenlylate transporter, as defined in claim 1.
  • the present invention includes and provides a nucleic acid molecule which comprises: (A) a promoter region which functions in a plant cell to cause the production of a mRNA molecule; (B) an exogenous structural nucleic acid molecule encoding a protein comprising an amino acid sequence of SEQ ID NO: 2; and (C) a 3' non-translated sequence that functions in the plant cell to cause termination of transcription and addition of polyadenylated ribonucleotides to a 3' end of the mRNA molecule, as reflected by claim 5.
  • the present invention includes and provides a nucleic acid molecule which comprises a substantially purified nucleic acid molecule comprising as operably linked components: (A) a promoter region which functions in a plant cell to cause the production of an mRNA molecule; (B) a nucleic acid sequence that encodes an adenylate transporter or a fragment there of; wherein the purified nucleic acid molecule further comprises one or more expression cassettes, each of which expresses a member selected from the group consisting of slr1736, ATPT2. dxs, dxr; GGH, GGPPS HPPD, MT1, TMT2, GMT, slr 1737, and an antisense construct for homogentisic acid dioxygenase.
  • the present invention includes and provides a transgenic plant comprising a substantially purified nucleic acid molecule comprising as operably linked components: (A) a promoter region which functions in a plant cell to cause the production of an mRNA molecule; (B) a nucleic acid sequence that encodes an adenylate transporter , wherein the purified nucleic acid molecule further comprises one or more expression cassettes, each of which expresses a member selected from the group consisting of slr1736 , ATPT2, dxs, dxr, GGH GGPPS, HPPD, MT1, TMT2, GMT, slr 1737 , and an antisense construct for homogentisic acid dioxygenase, as further specified in claim 6.
  • the present invention includes and provides a substantially purified protein comprising SEQ ID NO: 2.
  • the present invention includes and provides an antibody capable of specifically binding a substantially purified protein comprising SEQ ID NO: 2.
  • the present invention includes and provides a nucleic acid construct as defined by claim 7.
  • the present invention includes and provides a transformed plant as defined in claim 10.
  • the present invention includes and provides a method of producing a plant as defined in claim 14.
  • the present invention includes and provides a seed as defined in claim 16. that encodes for a protein having an amino acid sequence of SEQ ID NO: 2, wherein the seed has an increased tocopherol level relative to seeds from a plant having a similar genetic background but lacking the exogenous nucleic acid molecule.
  • the present invention includes and provides a seed derived from a transformed plant having an exogenous nucleic acid molecule comprising SEQ ID NO: 1, wherein the seed has an increased tocopherol level relative to seeds from a plant having a similar genetic background but lacking the exogenous nucleic acid molecule.
  • the present invention provides a number of agents, for example, nucleic acid molecules and proteins associated with adenylate translocation, and provides uses of such agents.
  • the agents of the invention will preferably be "biologically active" with respect to either a structural attribute, such as the capacity of a nucleic acid to hybridize to another nucleic acid molecule, or the ability of a protein to be bound by an antibody (or to compete with another molecule for such binding). Alternatively, such an attribute may be catalytic and thus involve the capacity of the agent to mediate a chemical reaction or response.
  • the agents will preferably be “substantially purified.”
  • the term “substantially purified,” as used herein, refers to a molecule separated from substantially all other molecules normally associated with it in its native state. More preferably a substantially purified molecule is the predominant species present in a preparation.
  • a substantially purified molecule may be greater than 60% free, preferably 75% free, more preferably 90% free, and most preferably 95% free from the other molecules (exclusive of solvent) present in the natural mixture.
  • the term "substantially purified” is not intended to encompass molecules present in their native state.
  • agents of the invention may also be "recombinant.”
  • the term “recombinant” means any agent (e.g ., DNA, peptide etc.), that is, or results from, however indirectly, human manipulation of a nucleic acid molecule.
  • a cell, organism, or plant may have a phenotype that is altered relative to a similar cell, organism, or plant having a "similar genetic background.”
  • a "similar genetic background” is a background where the organisms being compared share 50% or greater of their nuclear genetic material.
  • a similar genetic background is a background where the organisms being compared share 75% or greater, even more preferably 90% or greater, of their nuclear genetic material.
  • a similar genetic background is a background where the organisms being compared are plants, and the plants are isogenic except for any genetic material originally introduced using plant transformation techniques.
  • the agents of the invention may be labeled with reagents that facilitate detection of the agent (e.g., fluorescent labels, Prober et al., Science 238:336-340 (1987 ); Albarella et al., EP 144914 ; chemical labels, Sheldon et al., U.S. Patent 4,582,789 ; Albarella et al., U.S. Patent 4,563,417 ; modified bases, Miyoshi et al., EP 119448 ).
  • fluorescent labels e.g., fluorescent labels, Prober et al., Science 238:336-340 (1987 ); Albarella et al., EP 144914 ; chemical labels, Sheldon et al., U.S. Patent 4,582,789 ; Albarella et al., U.S. Patent 4,563,417 ; modified bases, Miyoshi et al., EP 119448 ).
  • Agents of the invention include nucleic acid molecules.
  • the nucleic acid molecules comprise a nucleic acid sequence which encodes an adenylate transporter.
  • an "adenylate transporter” is a protein with the amino acid set forth in SEQ ID NO: 2.
  • nucleic acid molecule comprises a nucleic acid sequence of SEQ ID NO: 1.
  • nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 2.
  • nucleic acids can encode a protein that differs from any of the proteins in that one or more amino acids have been deleted, substituted, or added without altering the function.
  • codons capable of coding for such conservative amino acid substitutions are known in the art.
  • fragment nucleic acid molecules may consist of significant portion(s) of, or indeed most of the nucleic acid molecules such as those specifically disclosed.
  • the fragments may comprise smaller oligonucleotides (having from about 15 to about 400 nucleotide residues and more preferably, about 15 to about 30 nucleotide residues, or about 50 to about 100 nucleotide residues, or about 100 to about 200 nucleotide residues, or about 200 to about 400 nucleotide residues, or about 275 to about 350 nucleotide residues).
  • One fragment nucleic acid molecule codes, at least in part, for a mitochondrial or plastid targeting peptide.
  • Another fragment nucleic acid molecule codes for a plastid targeting peptide.
  • a PCR probe is a nucleic acid molecule capable of initiating a polymerase activity while in a double-stranded structure with another nucleic acid.
  • amino acids in a native sequence can be substituted with other amino acid(s), the charge and polarity of which are similar to that of the native amino acid, i.e. , a conservative amino acid substitution.
  • Conservative substitutes for an amino acid within the native polypeptide sequence can be selected from other members of the class to which the amino acid belongs.
  • Amino acids can be divided into the following four groups: (1) acidic amino acids, (2) basic amino acids, (3) neutral polar amino acids, and (4) neutral, nonpolar amino acids.
  • amino acids within these various groups include, but are not limited to, (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.
  • amino acid substitution within the native polypeptide sequence can be made by replacing one amino acid from within one of these groups with another amino acid from within the same group.
  • biologically functional equivalents of the proteins or fragments thereof of the present invention can have ten or fewer conservative amino acid changes, more preferably seven or fewer conservative amino acid changes, and most preferably five or fewer conservative amino acid changes.
  • the encoding nucleotide sequence will thus have corresponding base substitutions, permitting it to encode biologically functional equivalent forms of the proteins or fragments of the present invention.
  • amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Because it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence and, of course, its underlying DNA coding sequence and, nevertheless, a protein with like properties can still be obtained. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the proteins or fragments of the present invention, or corresponding DNA sequences that encode said peptides, without appreciable loss of their biological utility or activity. It is understood that codons capable of coding for such amino acid changes are known in the art.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art ( Kyte and Doolittle, J Mol. Biol. 157, 105-132 (1982 )). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics ( Kyte and Doolittle, J. Mol. Biol. 157:105-132 (1982 )). These are isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine/cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamate (-3.5), glutamine (-3.5), aspartate (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5).
  • substitution of amino acids which have hydropathic indices within ⁇ 2 is preferred, those which have hydropathic indices within ⁇ 1 are particularly preferred, and those which have hydropathic indices within ⁇ 0.5 are even more particularly preferred.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0), lysine (+3.0), aspartate (+3.0 ⁇ 1), glutamate (+3.0 ⁇ 1), serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (-0.4), proline (-0.5 ⁇ 1), alanine (-0.5), histidine (-0.5), cysteine (-1.0), methionine (-1.3), valine (-1.5), leucine (-1.8), isoleucine (-1.8), tyrosine (-2.3), phenylalanine (-2.5), and tryptophan (-3.4).
  • substitution of amino acids which have hydrophilicity values within ⁇ 2 is preferred, those which have hydropathic indices within ⁇ 1 are particularly preferred, and those which have hydropathic indices within ⁇ 0.5 are even more particularly preferred.
  • a class of agents includes the protein as defined in claim 8.
  • protein or “peptide molecule” includes any molecule that comprises five or more amino acids. It is well known in the art that proteins may undergo modification, including post-translational modifications, such as, but not limited to, disulfide bond formation, glycosylation, phosphorylation, or oligomerization- Thus, as used herein, the term “protein” or “peptide molecule” includes any protein that is modified by any biological or non-biological process.
  • amino acid and “amino acids” refer to all naturally occurring L-amino acids. This definition is meant to include norleucine, norvaline, ornithine, homocysteine, and homoserine.
  • One or more of the protein or fragments thereof or peptide molecules may be produced via chemical synthesis, or more preferably, by expression in a suitable bacterial or eukaryotic host. Suitable methods for expression are described by Sambrook et al., In: Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold”Spring Harbor Press, Cold Spring Harbor, New York (1989 ) or similar texts.
  • a “protein fragment” is a peptide or polypeptide molecule whose amino acid sequence comprises a subset of the amino acid sequence of that protein.
  • a protein or fragment thereof that comprises one or more additional peptide regions not derived from that protein is a "fusion" protein.
  • Such molecules may be derivatized to contain carbohydrate or other moieties (such as keyhole limpet hemocyanin). Fusion proteins or peptide molecules of the invention are preferably produced via recombinant means.
  • Exogenous genetic material may be transferred into a plant cell and the plant cell regenerated into a whole, fertile or sterile plant.
  • Exogenous genetic material is any genetic material, whether naturally occurring or otherwise, from any source that is capable of being inserted into any organism.
  • the exogenous genetic material encodes a plant adenylate transporter, as defined in claims 1 and 8, respectively.
  • exogenous genetic material comprising the adenylate transporter is introduced into a plant with one or more additional genes.
  • preferred combinations of genes include two or more of the following genes: tyrA, slr1736, ATPT2, dxs, dxr, GGPPS, HPPD, GMT, AANT1, slr 1737, and an antisense construct for Homogentisic acid dioxygenase ( Kridl et al., Seed Sci. Res. 1:209:219 (1991 ); Keegstra, Cell 56(2):247-53 (1989 ); Nawrath, et al., Proc. Natl. Acad. Sci. U.S.A.
  • one or more of the gene products can be directed to the plastid by the use of a plastid targeting sequence.
  • one or more of the gene products can be localized in the cytoplasm.
  • genes can be introduced, for example, with the adenylate transporter homolog or fragment thereof and one or more additional genes on a single construct, or with the adenylate transporter homolog or fragment thereof and one or more additional genes introduced on different constructs but the with same transformation event, or introduced into separate plants followed by one or more crosses to generate the desired combination of genes.
  • a preferred promoter is a napin promoter and a preferred plastid targeting sequence is a CTP1 sequence.
  • DNA constructs can be generated as "multiple gene constructs".
  • the multiple gene constructs contain multiple genes each under the control of a separate promoter, for example a seed specific promoter such as the napin promoter, arcelin 5, or 7S promoter.
  • the products of each of the genes can be individually targeted to the plastid by an encoded plastid target peptide.
  • the multiple gene construct or constructs can have one or more of the following genes in addition to AANT1: tyrA, slr1736, ATPT2, dxs, dxr, GGPPS, HPPD, GMT, AANT1, slr 1737, MT1, TMT2, and an antisense construct for homogentisic acid dioxygenase.
  • Such genetic material may be transferred into either monocotyledons or dicotyledons including, but not limited to, maize, soybean, canola, Arabidopsis, phaseolus, peanut, alfalfa, wheat, rice, oat, sorghum, rye, tritordeum, millet, fescue, perennial ryegrass, sugarcane, cranberry, papaya, banana, safflower, oil palm, flax, muskmelon, apple, cucumber, dendrobium, gladiolus, chrysanthemum, liliacea, cotton, eucalyptus, sunflower, Brassica campestris, Brassica napus, turfgrass, sugarbeet, coffee, and dioscorea ( Christou, In: Particle Bombardment for Genetic Engineering of Plants, Biotechnology Intelligence Unit.
  • the genetic material is transferred into soybean.
  • the genetic material is transferred into canola.
  • the genetic material is transferred into Brassica napus.
  • Transfer of a nucleic acid that encodes a protein can result in expression or overexpression of that protein in a transformed cell or transgenic plant.
  • One or more of the proteins encoded by nucleic acid molecules of the invention may be overexpressed in a transformed cell or transformed plant. Such expression or overexpression may be the result of transient or stable transfer of the exogenous genetic material.
  • expression or overexpression of the protein of the present invention in a plant provides in that plant, relative to an untransformed plant with a similar genetic background, an increased level of tocopherols.
  • overexpression of the protein in a plant provides in that plant, relative to an untransformed plant with a similar genetic background, an increased level of ⁇ -tocopherols.
  • overexpression of the protein in a plant provides in that plant, relative to an untransformed plant with a similar genetic background, an increased level of ⁇ -tocopherols.
  • overexpression of the protein in a plant provides in that plant, relative to an untransformed plant with a similar genetic background, an increased level of homogentisic acid.
  • overexpression of the protein in a plant provides in that plant, relative to an untransformed plant with a similar genetic background, an increased level of phytylpyrophosphate acid.
  • overexpression of the protein in a transformed plant may provide tolerance to a variety of stressors, e.g, oxidative stress tolerance such as to oxygen or ozone, UV tolerance, cold tolerance, or fungal/microbial pathogen tolerance.
  • stressors e.g, oxygen or ozone, UV tolerance, cold tolerance, or fungal/microbial pathogen tolerance.
  • a tolerance or resistance, to stress is determined by the ability of a plant, when challenged by a stressor such as cold, to produce a plant having a higher yield than a plant without such tolerance or resistance to stress.
  • the tolerance or resistance to stress is measured relative to a plant with a similar genetic background to the tolerant or resistant plant except that the plant expresses or overexpresses a protein of fragment thereof of the present invention.
  • Exogenous genetic material may be transferred into a host cell by the use of a DNA vector or construct designed for such a purpose. Design of such a vector is generally within the skill of the art ( See, Plant Molecular Biology: A Laboratory Manual, Clark'(ed.), Springer, New York (1997 )).
  • DNA vector constructs may be multiple gene constructs that comprise an expession casset for the adenylate transporter or a fragment thereof and one or more gene sequences selected from the group consisting of tyrA, slr1736, ATPT2, dxs, dxr , GGH, GGPPS, HPPD, MT1, TMT2, GMT, AANT1, slr 1737, and an antisense construct for homogentisic acid dioxygenase, such that transformation with a single DNA vector construct will result in the expression of the two or more of the gene sequences.
  • a construct or vector may include a plant promoter to express the protein of choice.
  • a number of promoters that are active in plant cells have been described in the literature. These include the nopaline synthase (NOS) promoter ( Ebert et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:5745-5749 (1987 )), the octopine synthase (OCS) promoter (which is carried on tumor-inducing plasmids of Agrobacterium tumefaciens ), the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter ( Lawton et al., Plant Mol. Biol.
  • NOS nopaline synthase
  • OCS octopine synthase
  • CaMV cauliflower mosaic virus
  • the CaMV 35S promoter Odell et al., Nature 313:810-812 (1985 )
  • the figwort mosaic virus 35S-promoter which is the light-inducible promoter from the small subunit of ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO)
  • the Adh promoter Walker et al., Proc. Natl. Acad. Sci. (US.A.) 84:6624-6628 (1957 )
  • the sucrose synthase promoter Yang et al., Proc. Natl. Acad. Sci.
  • promoters utilized For the purpose of expression in source tissues of the plant, such as the leaf, seed, root or stem, it is preferred that the promoters utilized have relatively high expression in these specific tissues. Tissue-specific expression of a protein of the present invention is a particularly preferred embodiment. For this purpose, one may choose from a number of promoters for genes with tissue- or cell-specific or -enhanced expression. Examples of such promoters reported in the literature include the chloroplast glutamine synthetase GS2 promoter from pea ( Edwards et al., Proc. Natl. Acad Sci.
  • RbcS ribulose-1,5-bisphosphate carboxylase
  • promoters for the thylakoid membrane proteins from spinach psa D , psa F , psa E , PC, FNR, atp C , atp D , cab, rbc S.
  • Other promoters for the chlorophyll a/b-binding proteins may also be utilized in the invention, such as the promoters for Lhc B gene and Psb P gene from white mustard ( Sinapis alba; Kretsch et al., Plant Mol. Biol. 28:219-229 (1995 )).
  • the promoters utilized in the invention have relatively high expression in these specific tissues.
  • a number of promoters for genes with tuber-specific or tuber-enhanced expression are known, including the class I patatin promoter ( Bevan et al., EMBO J. 8:1899-1906 (1986 ); Jefferson et al., Plant Mol. Biol.
  • the promoter for the potato tuber ADPGPP genes both the large and small subunits, the sucrose synthase promoter ( Salanoubat and Belliard, Gene 60:47-56 (1987 ), Salanoubat and Belliard, Gene 84:181-185 (1989 )), the promoter for the major tuber proteins including the 22 kd protein complexes and protease inhibitors ( Hannapel, Plant Physio/. 101:703-704 (1993 )), the promoter for the granule-bound starch synthase gene (GBSS) ( Visser et al., Plant Mol. Biol.
  • promoters can also be used to express a protein or fragment thereof in specific tissues, such as seeds or fruits.
  • the promoter used is a seed specific promoter.
  • promoters include the 5' regulatory regions from such genes as napin ( Kridl et al., Seed Sci. Res. 1:209:219 (1991 )), phaseolin ( Bustos, et al., Plant Cell, 1(9):839-853 (1989 )), soybean trypsin inhibitor ( Riggs, et al., Plant Cell 1(6):609-621 (1989 )), ACP ( Baerson, et al., Plant Mol.
  • a further preferred promoter is the arcelin 5 (Arc5) promoter ( Goossens et al., Plant Physiol. 120:1095-1104 (1999 ), and as disclosed in US 2005/0041018 and US 2003/0046727 ).
  • zeins which are a group of storage proteins found in maize endosperm. Genomic clones for zein genes have been isolated ( Pedersen et al., Cell 29:1015-1026 (1982 ), and Russell et al., Transgenic Res. 6(2):157-168 ) and the promoters from these clones, including the 15 kD, 16 kD, 19 kD, 22 kD, 27 kD and genes, could also be used.
  • promoters known to function for example, in maize include the promoters for the following genes: waxy, Brittle, Shrunken 2, Branching enzymes I and II, starch synthases, debranching enzymes, oleosins, glutelins and sucrose synthases.
  • a particularly preferred promoter for maize endosperm expression is the promoter for the glutelin gene from rice, more particularly the Osgt-1 promoter ( Zheng et al., Mol. Cell Biol. 13:5829-SS42 (1993 )).
  • promoters suitable for expression in wheat include those promoters for the ADPglucose pyrosynthase (ADPGPP) subunits, the granule bound and other starch synthase, the branching and debranching enzymes, the embryogenesis-abundant proteins, the gliadins and the glutenins.
  • promoters in rice include those promoters for the ADPGPP subunits, the granule bound and other starch synthase, the branching enzymes, the debranching enzymes, sucrose synthases and the glutelins.
  • a particularly preferred promoter is the promoter for rice glutelin, Osgt-1.
  • promoters for barley include those for the ADPGPP subunits, the granule bound and other starch synthase, the branching enzymes, the debranching enzymes, sucrose synthases, the hordeins, the embryo globulins, and the aleurone specific proteins.
  • a preferred promoter for expression in the seed is a napin promoter.
  • Root specific promoters may also be used.
  • An example of such a promoter is the promoter for the acid chitinase gene ( Samac et al., Plant Mol. Biol. 25:587-596 (1994 )). Expression in root tissue could also be accomplished by utilizing the root specific subdomains of the CaMV35S promoter that have been identified ( Lam et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:7890-7894 (1989 )).
  • Other root cell specific promoters include those reported by Conkling et al. ( Conkling et al., Plant Physiol. 93:1203-1211 (1990 )).
  • Constructs or vectors may also include, with the coding region of interest, a nucleic acid sequence that acts, in whole or in part, to terminate transcription of that region.
  • a nucleic acid sequence that acts, in whole or in part, to terminate transcription of that region.
  • Regulatory transcript termination regions can be provided in plant expression constructs of this invention as well.
  • Transcript termination regions can be provided by the DNA sequence encoding the gene of interest or a convenient transcription termination region derived from a different gene source, for example, the transcript termination region which is naturally associated with the transcript initiation region. The skilled artisan will recognize that any convenient transcript termination region which is capable of terminating transcription in a plant cell can be employed in the constructs of the present invention.
  • a vector or construct may also include regulatory elements.
  • regulatory elements include the Adh intron 1 ( Callis et al., Genes and Develop. 1:1183-1200 (1987 )), the sucrose synthase intron ( Vasil et al., Plant Physiol. 91:1575-1579 (1989 )), and the TMV omega element ( Gallie et. al., The Plant Cell 1:301-311 (1989 )). These and other regulatory elements may be included when appropriate.
  • a vector or construct may also include a selectable marker.
  • Selectable markers may also be used to select for plants or plant cells that contain the exogenous genetic material. Examples of such include, but are not limited to: a neo gene ( Potrykus et al., Mol. Gen. Genet. 199:183-188 (1985 )) which codes for kanamycin resistance and can be selected for using kanamycin, RptII, G418, hpt, etc.; a bar gene which codes for bialaphos resistance; a mutant EPSP synthase gene ( Hinchee et al., Bio/Technology 6:915-922 (1988 ); Reynaerts et al., Selectable and Screenable Markers.
  • ALS acetolactate synthase gene
  • European Patent Application 154,204 Sept. 11, 1985
  • ALS D'Halluin et al., Bio/Technology 10: 309-314 (1992 )
  • methotrexate resistant DHFR gene Thillet et al., J. Biol. Chem. 263:12500-12508 (1988 )
  • a vector or construct may also include a transit peptide. Incorporation of a suitable chloroplast transit peptide may also be employed (European Patent Application Publication Number 0218571 ).
  • Translational enhancers may also be incorporated as part of the vector DNA.
  • DNA constructs could contain one or more 5' non-translated leader sequences which may serve to enhance expression of the gene products from the resulting mRNA transcripts. Such sequences may be derived from the promoter selected to express the gene or can be specifically modified to increase translation of the mRNA. Such regions may also be obtained from viral RNAs, from suitable eukaryotic genes, or from a synthetic gene sequence. For a review of optimizing expression of transgenes, see Koziel et al., Plant Mol. Biol. 32:393-405 (1996 ).
  • a preferred plastid transit peptide sequence is CTP1.
  • a vector or construct may also include a screenable marker.
  • Screenable markers may be used to monitor expression.
  • Exemplary screenable markers include: a ⁇ -glucuronidase or uid A gene (GUS) which encodes an enzyme for which various chromogenic substrates are known ( Jefferson, Plant Mol. Biol, Rep. 5:387-405 (1987 ); Jefferson et al., EMBO J.
  • tyrosinase gene ( Katz et al., J. Gen. Microbiol. 129:2703-2714 (1983 )) which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in turn condenses to melanin; and an ⁇ -galactosidase which will turn a chromogenic ⁇ -galactose substrate.
  • selectable or screenable marker genes are also genes which encode a secretable marker whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers which encode a secretable antigen that can be identified by antibody interaction, or even secretable enzymes which can be detected catalytically.
  • Secretable proteins fall into a number of classes, including small, diffusible proteins which are detectable, (e.g., by ELISA), small active enzymes which are detectable in extracellular solution (e.g., ⁇ -amylase, ⁇ -lactamase, phosphinothricin transferase), or proteins which are inserted or trapped in the cell wall (such as proteins which include a leader sequence such as that found in the expression unit of extension or tobacco PR-S).
  • small active enzymes which are detectable in extracellular solution
  • proteins which are inserted or trapped in the cell wall such as proteins which include a leader sequence such as that found in the expression unit of extension or tobacco PR-S.
  • proteins which include a leader sequence such as that found in the expression unit of extension or tobacco PR-S.
  • Other possible selectable and/or screenable marker genes will be apparent to those of skill in the art.
  • nucleic acid molecules may be introduced into a cell, such as by Agrobacterium infection or direct delivery of nucleic acid molecules such as, for example, by PEG-meditated transformation, by electroporation, by acceleration of DNA coated particles, etc.
  • Agrobacterium infection or direct delivery of nucleic acid molecules
  • electroporation has been used to transform maize protoplasts ( Fromm et al., Nature 312:791-793 (1986 )).
  • vector systems suitable for introducing transforming DNA into a host plant cell include but are not limited to binary artificial chromosome (BIBAC) vectors ( Hamilton et al., Gene 200:107-116 (1997 )); and transfection with RNA viral vectors ( Della-Cioppa et al., Ann. N.Y. Acad. Sci. (1996), 792 ( Engineering Plants for Commercial Products and Applications), 57-61 ). Additional vector systems also include plant selectable YAC vectors such as those described in Mullen et al., Molecular Breeding 4:449-457 (1988 ).
  • BIBAC binary artificial chromosome
  • Acceleration methods include, for example, microprojectile bombardment and the like.
  • microprojectile bombardment One example of a method for delivering transforming nucleic acid molecules into plant cells is microprojectile bombardment. This method has been reviewed by Yang and Christou (eds.), Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994 ).
  • Non-biological particles may be coated with nucleic acids and delivered into cells by a propelling force.
  • Exemplary particles include those comprised of tungsten, gold, platinum and the like.
  • a particular advantage of microprojectile bombardment in addition to it being an effective means of reproducibly transforming monocots, is that neither the isolation of protoplasts ( Cristou et al., Plant Physiol. 87:671-674 (1988 )) nor the susceptibility to Agrobacterium infection is required.
  • An illustrative embodiment of a method for delivering DNA into maize cells by acceleration is a biolistics ⁇ -particle delivery system, which can be used to propel particles coated with DNA through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with maize cells cultured in suspension.
  • Gordon-Kamm et al. describes the basic procedure for coating tungsten particles with DNA ( Gordon-Kamm et al., Plant Cell 2:603-618 (1990 )).
  • the screen disperses the tungsten nucleic acid particles so that they are not delivered to the recipient cells in large aggregates.
  • a particle delivery system suitable for use with the invention is the helium acceleration PDS-1000/He gun, which is available from Bio-Rad Laboratories (Bio-Rad, Hercules, California)( Sanford et al., Technique 3:3-16 (1991 )).
  • cells in suspension may be concentrated on filters.
  • Filters containing the cells to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate. If desired, one or more screens are also positioned between the gun and the cells to be bombarded.
  • immature embryos or other target cells may be arranged on solid culture medium.
  • the cells to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate.
  • one or more screens are also positioned between the acceleration device and the cells to be bombarded.
  • bombardment transformation one may optimize the pre-bombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants.
  • Both the physical and biological parameters for bombardment are important in this technology. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the flight and velocity of either the macro- or microprojectiles.
  • Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids. It is believed that pre-bombardment manipulations are especially important for successful transformation of immature embryos.
  • plastids can be stably transformed.
  • Methods disclosed for plastid transformation in higher plants include the particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination ( Svab et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:8526-8530 (1990 ); Svab and Maliga, Proc. Natl. Acad. Sci. (U.S.A.) 90:913-917 (1993 ); Staub and Maliga, EMBO J. 12:601-606 (1993 ); U.S. Patents 5, 451,513 and 5,545,818 ).
  • the execution of other routine adjustments will be known to those of skill in the art in light of the present disclosure.
  • Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact.plant from a protoplast.
  • the use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art. See, for example the methods described by Fraley et al., Bio/Technology 3:629-635 (1985 ) and Rogers et al., Methods Enzymol. 153:253-277 (1987 ). Further, the integration of the Ti-DNA is a relatively precise process resulting in few rearrangements.
  • the region of DNA to be transferred is defined by the border sequences and intervening DNA is usually inserted into the plant genome as described ( Spielmann et al., Mol. Gen. Genet. 205:34 (1986 )).
  • Modem Agrobacterium transformation vectors are capable of replication in E . coli as well as Agrobacterium, allowing for convenient manipulations as described ( Klee et al., In: Plant DNA Infectious Agents, Hohn and Schell (eds.), Springer-Verlag, New York, pp. 179-203 (1985 )). Moreover, technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expressing various polypeptide coding genes.
  • the vectors described have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes ( Rogers et al., Methods Enzymol. 153:253-277 (1997 )).
  • Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant strains where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.
  • a transgenic plant formed using Agrobacterium transformation methods typically contains a single gene on one chromosome. Such transgenic plants can be referred to as being heterozygous for the added gene. More preferred is a transgenic plant that is homozygous for the added structural gene; i.e., a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair.
  • a homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant, transgenic plant that contains a single added gene, germinating some of the seed produced, and analyzing the resulting plants produced for the gene of interest.
  • transgenic plants can also be mated to produce offspring that contain two independently segregating, exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both added, exogenous genes that encode a polypeptide of interest. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation.
  • Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation and combinations of these treatments ( See, for example, Potrykus et al., Mol. Gen. Genet. 205:193-200 (1986 ); Lorz et al., Mol. Gen. Genet. 199:178 (1985 ); Fromm et al., Nature 319:791 (1956 ); Uchimiya et al., Mol. Gen. Genet. 204:204 (1986 ); Marcotte et al., Nature 335:454-457 (1988 )).
  • Other methods of cell transformation can also be used and include but are not limited to introduction of DNA into plants by direct DNA transfer into pollen ( Hess et al., Intern Rev. Cytol. 107:367 (1987 ); Luo et al., Plant Mol Biol. Reporter 6:165 (1988 )), by direct injection of DNA into reproductive organs of a plant ( Pena et al., Nature 325:274 (1987 )), or by direct injection of DNA into the cells of immature embryos followed by the rehydration of desiccated embryos ( Neuhaus et al., Then. Appl. Genet. 75:30 (1987 )).
  • This regeneration and growth process typically includes the steps of selection of transformed cells and culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
  • the development or regeneration of plants containing the foreign, exogenous gene that encodes a protein of interest is well known in the art.
  • the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed with seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants.
  • a transgenic plant of the invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art.
  • Transformation of monocotyledons using electroporation, particle bombardment, and Agrobacterium have also been reported. Transformation and plant regeneration have been achieved in asparagus ( Bytebier et al., Proc. Natl. Acad. Sci. (USA) 84:5354 (1987 )); barley ( Wan and Lemaux, Plant Physiol 104:37 (1994 )); maize ( Rhodes et al., Science 240:204 (1988 ); Gordon-Kamm et al., Plant Cell 2:603-618 (1990 ); Fromm et al., Bio/Technology 8:833 (1990 ); Koziel et al., Bio/Technology 11:194 (1993 ); Armstrong et al., Crop Science 35: 550-557 (1995 )); oat ( Somers et al., Bio/Technology 10:1589 (1992 )); orchard grass ( Horn et al., Plant Cell Rep.
  • a nucleic acid molecule of the invention may be introduced into a plant cell in a permanent or transient manner in combination with other genetic elements such as vectors, promoters, enhancers, etc. Further, the nucleic acid molecule of the invention may be introduced into a plant cell in a manner that allows for expression or overexpression of the protein encoded by the nucleic acid molecule.
  • Cosuppression is the reduction in expression levels, usually at the level of RNA, of a particular endogenous gene or gene family by the expression of a homologous sense construct that is capable of transcribing mRNA of the same strandedness as the transcript of the endogenous gene ( Napoli et al., Plant Cell 2:279-289 (1990 ); van der Krol et al., Plant Cell 2:291-299 (1990 )). Cosuppression may result from stable transformation with a single copy nucleic acid molecule that is homologous to a nucleic acid sequence found within the cell ( Prolls and Meyer, Plant J.
  • Antisense approaches are a way of preventing or reducing gene function by targeting the genetic material ( Mol et al., FEBS Lett 268:427-430 (1990 )).
  • the objective of the antisense approach is to use a sequence complementary to the target gene to block its expression and create a mutant cell line or organism in which the level of a single chosen protein is selectively reduced or abolished.
  • Antisense techniques have several advantages over other reverse genetic' approaches. The site of inactivation and its developmental effect can be manipulated by the choice of promoter for antisense genes or by the timing of external application or microinjection.
  • Antisense can manipulate its specificity by selecting either unique regions of the target gene or regions where it shares homology to other related genes ( Hiatt et al., In: Genetic Engineering, Setlow (ed.), Vol. 11, New York: Plenum 49-63 (1989 )).
  • Antisense RNA techniques involve introduction of RNA that is complementary to the target RNA into cells, which results in specific RNA:RNA duplexes being formed by base pairing between the antisense substrate and the target mRNA ( Green et al., Annu. Rev. Biochem. 55:569-597 (1986 )).
  • the process involves the introduction and expression of an antisense gene sequence.
  • Such a sequence is one in which part or all of the normal gene sequences are placed under a promoter in inverted orientation so that the 'wrong' or complementary strand is transcribed into a noncoding antisense RNA that hybridizes with the target mRNA and interferes with its expression ( Takayama and Inouye, Crit. Rev. Biochem. Mol. Biol.
  • An antisense vector is constructed by standard procedures and introduced into cells by transformation, transfection, electroporation, microinjection, infection, etc. The type of transformation and choice of vector will determine whether expression is transient or stable.
  • the promoter used for the antisense gene may influence the level, timing, tissue, specificity, or inducibility of the antisense inhibition.
  • the activity of a protein in a plant cell may be reduced or depressed by growing a transformed plant cell containing a nucleic acid molecule whose non-transcribed strand encodes a protein or fragment thereof.
  • Posttranscriptional gene silencing can result in virus immunity or gene silencing in plants.
  • PTGS is induced by dsRNA and is mediated by an RNA-dependent RNA polymerase, present in the cytoplasm, that requires a dsRNA template.
  • the dsRNA is formed by hybridization of complementary transgene mRNAs or complementary regions of the same transcript. Duplex formation can be accomplished by using transcripts from one sense gene and one antisense gene collocated in the plant genome, a single transcript that has self-complementarity, or sense and antisense transcripts from genes brought together by crossing.
  • the dsRNA-dependent RNA polymerase makes a complementary strand from the transgene mRNA and RNAse molecules attach to this complementary strand (cRNA).
  • cRNA complementary strand
  • These cRNA-RNase molecules hybridize to the endogene mRNA and cleave the single-stranded RNA adjacent to the hybrid.
  • the cleaved single-stranded RNAs are further degraded by other host RNAses because one will lack a capped 5' end and the other will lack a poly(A) tail ( Waterhouse et al., PNAS 95: 13959-13964 (1998 )).
  • nucleic acid of the invention may be introduced into a plant cell and transcribed using an appropriate promoter with such transcription resulting in the postranscriptional gene silencing of an endogenous transcript.
  • Antibodies have been expressed in plants ( Hiatt et al., Nature 342: 76-78 (1989 ); Conrad and Fielder, Plant Mol. Biol. 26: 3023-1030 (1994 )). Cytoplasmic expression of an scFv (single-chain Fv antibody) has been reported to delay infection by artichoke mottled crinkle virus. Transgenic plants that express antibodies directed against endogenous proteins may exhibit a physiological effect ( Philips et al., EMBO J. 16:4489-4496 (1997 ); Marion-Poll, Trends in Plant Science 2:447-448 (1997 )). For example, expressed anti-abscisic antibodies have been reported to result in a general perturbation of seed development ( Philips et al., EMBO J. 16: 4489-4496 (1997 )).
  • Antibodies that are catalytic may also be expressed in plants (abzymes).
  • abzymes The principle behind abzymes is that since antibodies maybe raised against many molecules, this recognition ability can be directed toward generating antibodies that bind transition states to force a chemical reaction forward ( Persidas; Nature Biotechnology 15: 1313-1315 (1997 ); Baca et al., Ann. Rev.Biophys Biomol. Struct. 26:461-493 (1997 )).
  • the catalytic abilities of abzymes may be enhanced by site directed mutagenesis. Examples of abzymes are, for example, set forth in U.S.Patent No: 5,658,753 ; U.S. Patent No. 5,632,990 ; U.S. Patent No: 5,631,137 ; U.S.
  • Patent 5,602,015 U.S. Patent No. 5;559,538 ; U.S. Patent No. 5,576,174 ; U.S: Patent No. 5,500,358 ; U.S. Patent 5,318,897 ; U.S. Patent No. 5,298,409 ; U.S. Patent No. 5,258,289 and U.S. Patent No,5,194,585 .
  • the antibody of the invention may be expressed in plants and that such expression can result in a physiological effect It is also understood that it may be catalytic.
  • the present invention also provides for parts of the plants. Such plant parts are seeds.
  • the seed is a constituent of animal feed.
  • Any of the plants or seeds thereof of the present invention may be processed to produce a feed, meal, protein, or oil-preparation.
  • the feed, meal, protein, or oil preparation is designed for ruminant animals.
  • Methods to produce feed, meal, protein, preparations are known in the art. See, for example, U.S. Patents 4,957,748 , 5,100,679 , 5,219,596 , 5,936,069 , 6,005,076 , 6,146,669 , and 6,156,227 .
  • the protein preparation is a high protein preparation.
  • Such a high protein preparation preferably has a protein content of at least about than 5% w/v, more preferably at least about 10% w/v, and even more preferably at least about 15% w/v.
  • the oil preparation is a high oil preparation with an oil content derived from a plant or part thereof of the present invention of at least about 5% w/v, more preferably at least about 10% w/v, and even more preferably at least about 15% w/v.
  • the oil preparation can be a liquid and of a volume greater than 1, 5, 10 or 50 liters.
  • oil produced from plants of the present invention or generated by a method of the present invention Such oil may be a minor or major component of any resultant product Moreover such oil may be blended with other oils.
  • the oil produced from plants of the present invention or generated by a method of the present invention constitutes greater than 0.5%, 1%, 5%, 10%, 25%, 50%, 75%, or 90% by volume or weight of the oil component of any product.
  • the oil preparation may be blended and can constitute greater than 10%, 25%, 35%, 50%, or 75% of the blend by volume.
  • Oil produced from a plant of the present invention can be admixed with one or more organic solvents or petroleum distillates.
  • Plants of the present invention can be part of or generated from a breeding program.
  • the choice of breeding method depends on the mode of plant reproduction, the heritability of the trait(s) being improved, and the type of cultivar used commercially (e.g. , F 1 hybrid cultivar, pureline cultivar, etc). Selected, non-limiting approaches for breeding the plants of the present invention are set forth below.
  • a breeding program can be enhanced using marker assisted selection of the progeny of any cross. It is further understood that any commercial and non-commercial cultivars can be utilized in a breeding program. Factors such as, for example, emergence vigor, vegetative vigor, stress tolerance, disease resistance, branching, flowering, seed set, seed size, seed density, standability, threshability, etc. will generally dictate the choice.
  • breeding method can be used to transfer one or a few favorable genes for a highly heritable trait into a desirable cultivar. This approach has been used extensively for breeding disease-resistant cultivars.
  • Various recurrent selection techniques are used to improve quantitatively inherited traits controlled by numerous genes. The use of recurrent selection in self-pollinating crops depends on the ease of pollination, the frequency of successful hybrids from each pollination, and the number of hybrid offspring from each successful cross.
  • Breeding lines can be tested and compared to appropriate standards in environments representative of the commercial target area(s) for two or more generations. The best lines are candidates for new commercial cultivars; those still deficient in traits may be used as parents to produce new populations for further selection.
  • One method of identifying a superior plant is to observe its performance relative to other experimental plants and to a widely grown standard cultivar. If a single observation is inconclusive, replicated observations can provide a better estimate of its genetic worth. A breeder can select and cross two or more parental lines, followed by repeated selfmg and selection, producing many new genetic combinations.
  • hybrid seed can be produced by manual crosses between selected male-fertile parents or by using male sterility systems.
  • Hybrids are selected for certain single gene traits such as pod color, flower color, seed yield, pubescence color, or herbicide resistance, which indicate that the seed is truly a hybrid. Additional data on parental lines, as well as the phenotype of the hybrid, influence the breeder's decision whether to continue with the specific hybrid cross.
  • Pedigree breeding and recurrent selection breeding methods can be used to develop cultivars from breeding populations. Breeding programs combine desirable traits from two or more cultivars or various broad-based sources into breeding pools from which cultivars are developed by selfing and selection of desired phenotypes. New cultivars can be evaluated to determine which have commercial potential.
  • Pedigree breeding is used commonly for the improvement of self-pollinating crops. Two parents who possess favorable, complementary traits are crossed to produce an F 1 population. An F 2 population is produced by selfing one or several members of the F 1 population. Selection of the best individuals from the best families is carried out. Replicated testing of families can begin in the F 4 generation to improve the effectiveness of selection for traits with low heritability. At an advanced stage of inbreeding (i.e., F 6 and F 7 ), the best lines or mixtures of phenotypically similar lines are tested for potential release as new cultivars.
  • Backcross breeding has been used to transfer genes for a simply inherited, highly heritable trait into a desirable homozygous cultivar or inbred line, which is the recurrent parent.
  • the source of the trait to be transferred is called the donor parent.
  • the resulting plant is expected to have the attributes of the recurrent parent (e.g. , cultivar) and the desirable trait transferred from the donor parent.
  • individuals possessing the phenotype of the donor parent are selected and repeatedly crossed (backcrossed) to the recurrent parent.
  • the resulting parent is expected to have the attributes of the recurrent parent (e.g. , cultivar) and the desirable trait transferred from the donor parent.
  • the single-seed descent procedure in the strict sense refers to planting a segregating population, harvesting a sample of one seed per plant, and using the one-seed sample to plant the next generation.
  • the plants from these lines are derived will each trace to different F 2 individuals.
  • the number of plants in a population declines each generation due to failure of some seeds to germinate or of some plants to produce at least one seed. As a result, not all of the F 2 plants originally sampled in the population will be represented by a progeny when generation advance is completed.
  • breeders commonly harvest one or more pods from each plant in a population and thresh them together to form a bulk. Part of the bulk is used to plant the next generation and part is put in reserve.
  • the procedure has been referred to as modified single-seed descent or the pod-bulk technique.
  • the multiple-seed procedure has been used to save labor at harvest. It is considerably faster to thresh pods with a machine than to remove one seed from each by hand for the single-seed procedure.
  • the multiple-seed procedure also makes it possible to plant the same number of seed of a population during each generation of inbreeding.
  • a transgenic plant of the present invention may also be reproduced using apomixis.
  • Apomixis is a genetically controlled method of reproduction in plants where the embryo is formed without union of an egg and a sperm.
  • pseudogamy or fertilization of the polar nuclei to produce endosperm is necessary for seed viability.
  • a nurse cultivar can be used as a pollen source for endosperm formation in seeds.
  • the nurse cultivar does not affect the genetics of the aposporous apomictic cultivar since the unreduced egg of the cultivar develops parthenogenetically, but makes endosperm production possible.
  • Apomixis is economically important, especially in transgenic plants, because it causes any genotype, no matter how heterozygous, to breed true.
  • heterozygous transgenic plants can maintain their genetic fidelity throughout repeated life cycles.
  • the nucleic acid of the present invention may be introduced into any cell or organism such as a mammalian cell, mammal, fish cell, fish, bird cell, bird, algae cell, algae, fungal cell, fungi, or bacterial cell.
  • the protein of the present invention may be produced in an appropriate cell or organism.
  • Preferred host and transformants include: fungal cells such as Aspergillus, yeasts, mammals, particularly bovine and porcine, insects, bacteria, and algae. Methods to transform such cells or organisms are known in the art ( EP 0 238 023 ; Yelton et al.. Proc Natl. Acad Sci.
  • overexpression of the protein in a cell or organism provides in that cell or organism, relative to an untransformed cell or organism with a similar genetic background, an increased level of tocopherols.
  • overexpression of the protein in a cell or organism provides in that cell or organism, relative to an untransformed cell or organism with a similar genetic background, an increased level of ⁇ -tocopherols. .
  • overexpression of the protein in a cell or organism provides in that cell or organism, relative to an untransformed cell or organism with a similar genetic background, an increased level of ⁇ -tocopherols.
  • overexpression of the protein in a cell or organism provides in that plant, relative to an untransformed plant with a similar genetic background, an increased level of homogentisic acid.
  • overexpression of the protein in a cell or organism provides in that plant, relative to an untransformed plant with a similar genetic background, an increased level of phytylpyrophosphate acid.
  • One aspect of the invention concerns the antibody of claim 9.
  • Such antibodies may be used to quantitatively or qualitatively detect the protein or peptide molecules of the invention.
  • an antibody or peptide is said to "specifically bind" to a protein or peptide molecule of the invention if such binding is not competitively inhibited by the presence of non-related molecules.
  • Nucleic acid molecules that encode the protein of the invention can be expressed, via recombinant means, to yield protein or peptides that can in turn be used to elicit antibodies that are capable of binding the expressed protein or peptide. Such antibodies may be used in immunoassays for that protein.
  • Such protein-encoding molecules may be a "fusion" molecule ( i.e ., a part of a larger nucleic acid molecule) such that, upon expression, a fusion protein is produced. It is understood that any of the nucleic acid molecules of the invention may be expressed, via recombinant means, to yield proteins or peptides encoded by these nucleic acid molecules.
  • the antibodies of the invention may be polyclonal or monoclonal and may comprise intact immunoglobulins, or antigen binding portions of immunoglobulin fragments (such as (F(ab'), F(ab') 2 ), or single-chain immunoglobulins producible, for example, via recombinant means. It is understood that practitioners are familiar with the standard resource materials which describe specific conditions and procedures for the construction, manipulation, and isolation of antibodies (see, for example, Harlow and Lane, In: Antibodies: A Laboratory MAnual, Cold Spring Harbor Press, Cold Spring Harbor, New York (1988 )).
  • antibody molecules or their fragments may be used for diagnostic purposes.
  • a ligand group such as biotin
  • a detectable marker group such as a fluorescent group, a radioisotope, or an enzyme
  • the ability to produce antibodies that bind the protein or peptide molecules of the invention permits the identification of mimetic compounds derived from those molecules. These mimetic compounds may contain a fragments of the protein or peptide or merely a structurally similar region and nonetheless exhibits an ability to specifically bind to antibodies directed against that compound.
  • Nucleic acid molecules of the invention may be employed to obtain other nucleic acid molecules from the same species (nucleic acid molecules from maize may be utilized to obtain other nucleic acid molecules from maize).
  • Such nucleic acid molecules include the nucleic acid molecules that encode the complete coding sequence of a protein and promoters and flanking sequences of such molecules.
  • nucleic acid molecules include nucleic acid molecules that encode other isozymes or gene family members. Such molecules can be readily obtained buy using the above-described nucleic acid molecules or fragments thereof to screen cDNA or genomic libraries. Methods for forming such libraries are well known in the art.
  • Nucleic acid molecules of the invention may also be employed to obtain nucleic acid homologs.
  • Such homolog include the nucleic acid molecules of other plants or other organisms (e.g., alfalfa, Arabidopsis, barley, Brassica, broccoli, cabbage, canola, citrus, cotton, garlic, oat, oilseed rape, onion, Brassica campestris, Brassica napus, flax, an ornamental plant, pea, peanut, pepper, potato, rice, rye, sorghum, strawberry, sugarcane, sugarbeet, tomato, wheat, poplar, pine, fir, eucalyptus, apple, lettuce, lentils, grape, banana, tea, turf grasses, sunflower, oil palm, maize, soybean, Phaseolus, etc.) including the nucleic acid molecules that encode, in whole or in part, protein homologues of other plant species or other organisms and sequences of genetic elements, such as promoters and transcriptional regulatory elements.
  • Particularly preferred plants are selected from the group consisting of canola, maize, Brassica campestris, Brassica napus, soybean, crambe, mustard, castor bean, peanuts sesame, cottonseed, linseed, safflower, oil palm, flax and sunflower.
  • Such molecules can be readily obtained by using the above-described nucleic acid molecules or fragments thereof to screen cDNA or genomic libraries obtained from such plant species. Methods for forming such libraries are well known in the art.
  • Such homolog molecules may differs in their nucleotide sequences from those found in SEQ ID NO: 1 or complements thereof because complete complementarity is not needed for stable hybridization.
  • any of a variety of methods may be used to obtain one or more of the above-described nucleic acid molecules ( Zamechik et aL, Proc. Natl. Acad Sci. (U.S.A.) 83:4143-4146 (1986 ); Goodchild et al., Proc:-Natl. Acad Sci. (U.S.A.) 85:5507-5511 (1988 ); Wickstrom et al., Proc. Natl. Acad Sci. (U.S.A.) 85:1028-1032 (1988 ); Holt et al., Molec. Cell. Biol.
  • nucleic acid synthesizers may be employed for this purpose.
  • the disclosed nucleic acid molecules may be used to define a pair of primers that can be used with the polymerase chain reaction ( Mullis et al., Cold Spring Harbor Symp. Quant. Biol.
  • Promoter sequences and other genetic elements, including but not limited to transcriptional regulatory flanking sequences, associated with one or more of the disclosed nucleic acid sequences can also be obtained using the disclosed nucleic acid sequence provided herein.
  • such sequences are obtained by incubating nucleic acid molecules of the present invention with members of genomic libraries and recovering clones that hybridize to such nucleic acid molecules thereof.
  • methods of "chromosome walking," or inverse PCR may be used to obtain such sequences ( Frohman et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:8998-9002 (1988 ); Ohara et al., Proc. Natl. Acad Sci.
  • chromosome walking means a process of extending a genetic map by successive hybridization steps.
  • the nucleic acid molecules of the invention may be used to isolate promoters of cell enhanced, cell specific, tissue enhanced, tissue specific, developmentally regulated, or environmentally regulated expression profiles. Isolation and functional analysis of the 5' flanking promoter sequences of these genes from genomic libraries, for example, using genomic screening methods and PCR techniques, would result in the isolation of useful promoters and transcriptional regulatory elements. These methods are known to those of skill in the art and have been described ( See, for example, Birren et al., Genome Analysis: Analyzing DNA, 1, (1997), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y .). Promoters obtained utilizing the nucleic acid molecules of the invention could also be modified to affect their control characteristics. Examples of such modifications would include, but are not limited to, enhancer sequences. Such genetic elements could be used to enhance gene expression of new and existing traits for crop improvement.
  • nucleic acid molecules of the invention includes nucleic acid molecules that are markers.
  • the markers can be used in a number of conventional ways in the field of molecular genetics.
  • Such markers include nucleic acid molecules of SEQ ID NO: 1, and complements thereof and fragments of either that can act as markers and other nucleic acid molecules of the present invention that can act as markers.
  • Genetic markers of the invention include "dominant” or “codominant” markers.
  • “Codominant markers” reveal the presence of two or more alleles (two per diploid individual) at a locus.
  • "Dominant markers” reveal the presence of only a single allele per locus.
  • the presence of the dominant marker phenotype e.g., a band of DNA).is an indication that one allele is in either the homozygous or heterozygous condition.
  • the absence of the dominant marker phenotype e.g. , absence of a DNA band
  • dominant and codominant markers can be equally valuable.
  • Marker molecules can be, for example, capable of detecting polymorphisms such as single nucleotide polymorphisms (SNPs).
  • SNPs single nucleotide polymorphisms
  • a "polymorphism” is a variation or difference in the sequence of the gene or its flanking regions that arises in some of the members of a species.
  • the variant sequence and the "original" sequence co-exist in the species' population. In some instances, such coexistence is in stable or quasi-stable equilibrium.
  • a polymorphism is thus said to be "allelic,” in that, due to the existence of the polymorphism, some members of a population may have the original sequence (i.e., the original "allele") whereas other members may have the variant sequence (i.e., the variant "allele”). In the simplest case, only one variant sequence may exist and the polymorphism is thus said to be di-allelic. In other cases, the species' population may contain multiple alleles and the polymorphism is termed tri-allelic, etc.
  • a single gene may have multiple different unrelated polymorphisms. For example, it may have a di-allelic polymorphism at one site and a multi-allelic polymorphism at another site.
  • the variation that defines the polymorphism may range from a single nucleotide variation to the insertion or deletion of extended regions within a gene.
  • the DNA sequence variations are in regions of the genome that are characterized by short tandem repeats (STRs) that include tandem di- or tri-nucleotide repeated motifs of nucleotides.
  • STRs short tandem repeats
  • Polymorphisms characterized by such tandem repeats are referred to as "variable number tandem repeat" ("VNTR") polymorphisms.
  • VNTRs have been used in identity analysis ( Weber, U.S. Patent 5,075,217 ; Armour et al., FEBS Lett. 307:113-115 (1992 ); Jones et al., Eur. J. Haematol.
  • the detection of polymorphic sites in a sample of DNA may be facilitated through the use of nucleic acid amplification methods. Such methods specifically increase the concentration of polynucleotides that span the polymorphic site, or include that site and sequences located either distal or proximal to it. Such amplified molecules can be readily detected by gel electrophoresis or other means.
  • such polymorphisms can be detected through the use of a marker nucleic acid molecule that is physically linked to such polymorphism(s).
  • marker nucleic acid molecules comprising a nucleotide sequence of a polynucleotide located within 1 mb of the polymorphism(s) and more preferably within 100kb of the polymorphism(s) and most preferably within 10kd of the polymorphism(s) can be employed.
  • the identification of a polymorphism can be determined in a variety of ways. By correlating the presence or absence of the polymorphism in a plant with the presence or absence of a phenotype, it is possible to predict the phenotype of that plant. If a polymorphism creates or destroys a restriction endonuclease cleavage site, or if it results in the loss or insertion of DNA (e.g., a VNTR polymorphism), it will alter the size or profile of the DNA fragments that are generated by digestion with that restriction endonuclease. As such, organisms that possess a variant sequence can be distinguished from those having the original sequence by restriction fragment analysis.
  • RFLPs restriction fragment length polymorphisms
  • Glassberg UK Patent Application 2135774 ; Skolnick et al., Cytogen. Cell Genet. 32:58-67 (1952 ); Botstein et al., Ann. J. Hum. Genet. 32:314-331 (1980 ); Fischer et al., (PCT Application WO90/13668 ; Uhlen, PCT Application WO90/11369 ).
  • Polymorphisms can also be identified by Single Strand Conformation Polymorphism (SSCP) analysis ( Elles, Methods in Molecular Medicine: Molecular Diagnosis of Genetic Diseases, Humana Press (1996 ); Orita et al., Genomics 5:874-879 (1989 )).
  • SSCP Single Strand Conformation Polymorphism
  • a number of protocols have been described for SSCP including, but not limited to, Lee et al., Anal. Biochem. 205:289-293 (1992 ); Suzuki et al., Anal. Biochem. 192: 82 84 (1991 ); Lo et al., Nucleic Acids Research 20:1005-1009 (1992 ); Sarkar et al., Genomics 13:441-443 (1992 ). It is understood that one or more of the nucleic acids of the invention may be utilized as markers or probes to detect polymorphisms by SSCP analysis.
  • Polymorphisms may also be found using a DNA fingerprinting technique called amplified fragment length polymorphism (AFLP), which is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA to profile that DNA ( Vos et al., Nucleic Acids Res. 23:4407-4414 (1995 )). This method allows for the specific co-amplification of high numbers of restriction fragments, which can be visualized by PCR without knowledge of the nucleic acid sequence. It is understood that one or more of the nucleic acids of the invention may be utilized as a marker or probe to detect polymorphisms by AFLP analysis or for fingerprinting RNA.
  • AFLP amplified fragment length polymorphism
  • Polymorphisms may also be found using random amplified polymorphic DNA (RAPD) ( Williams et al., Nucl. Acids Res. 18:6531-6535 (1990 )) and cleaveable amplified polymorphic sequences (CAPS) ( Lyamichev et al., Science 260:778-783 (1993 )). It is understood that one or more of the nucleic acid molecules of the invention may be utilized as markers or probes to detect polymorphisms by RAPD or CAPS analysis.
  • RAPD random amplified polymorphic DNA
  • CAS cleaveable amplified polymorphic sequences
  • Single Nucleotide Polymorphisms generally occur at a greater frequency than other polymorphic markers and are spaced with a greater uniformity throughout a genome than other reported forms of polymorphism.
  • the greater frequency and uniformity of SNPs means that there is greater probability that such a polymorphism will be found near or in a genetic locus of interest than would be the case for other polymorphisms.
  • SNPs are located in protein-coding regions and noncoding regions of a genome. Some of these SNPs may result in defective or variant protein expression (e.g., as a result of mutations or defective splicing). Analysis (genotyping) of characterized SNPs can require only a plus/minus assay rather than a lengthy measurement, permitting easier automation.
  • SNPs can be characterized using any of a variety of methods. Such methods include the direct or indirect sequencing of the site, the use of restriction enzymes ( Botstein et al., Am J. Hum. Genet. 32:314-331 (1980 ); Konieczny and Ausubel, Plant J. 4:403-410 (1993 )), enzymatic and chemical mismatch assays ( Myers et al., Nature 313:495-498 (1985 )), allele-specific PCR ( Newton et al., Nucl. Acids Res. 17:2503-2516 (1989 ); Wu et al., Proc. Natl. Acad Sci.
  • restriction enzymes Botstein et al., Am J. Hum. Genet. 32:314-331 (1980 ); Konieczny and Ausubel, Plant J. 4:403-410 (1993 )
  • enzymatic and chemical mismatch assays Myers et al., Nature 313:495-4
  • Polymorphisms may also be detected using allele-specific oligonucleotides (ASO), which can be, for example, used in combination with hybridization based technology including Southern, northern, and dot blot hybridizations, reverse dot blot hybridizations and hybridizations performed on microarray and related technology.
  • ASO allele-specific oligonucleotides
  • the stringency of hybridization for polymorphism detection is highly dependent upon a variety of factors, including length of the allele-specific oligonucleotide, sequence composition, degree of complementarity (i.e. presence or absence of base mismatches), concentration of salts and other factors such as formamide and temperature. These factors are important both during the hybridization itself and during subsequent washes performed to remove target polynucleotides that are not specifically hybridized. In practice, the conditions of the final, most stringent wash are most critical.
  • the amount of target polynucleotide that is able to hybridize to the allele-specific, oligonucleotide is also governed by such factors as the concentration of both the ASO and the target polynucleotide, the presence and concentration of factors that act to "tie up" water molecules so as to effectively concentrate the reagents (e.g. , PEG, dextran, dextran sulfate, etc .) whether the nucleic acids are immobilized or in solution, and the duration of hybridization and washing steps.
  • the reagents e.g. , PEG, dextran, dextran sulfate, etc .
  • Hybridizations are preferably performed below the melting temperature (T m ) of the ASO.
  • T m melting temperature
  • Other formulas for approximating T m are available and are known to those of ordinary skill in the art.
  • Stringency is preferably adjusted so as to allow a given ASO to differentially hybridize to a target polynucleotide of the correct allele and a target polynucleotide of the incorrect allele.
  • there will be at least a two-fold differential between the signal produced by the ASO hybridizing to a target polynucleotide of the correct allele and the level of the signal produced by the ASO cross-hybridizing to a target polynucleotide of the incorrect allele e.g. , an ASO specific for a mutant allele cross-hybridizing to a wild-type allele.
  • Requirements for marker-assisted selection in a plant breeding program are: (1) the marker(s) should co-segregate or be closely linked with the desired trait; (2) an efficient means of screening large populations for the molecular marker(s) should be available; and (3) the screening technique should have high reproducibility across laboratories and preferably be economical to use and user-friendly.
  • the genetic linkage of marker molecules can be established by a gene mapping model such as, without limitation, the flanking marker model reported by Lander and Botstein, Genetics 121:185-199 (1989 ) and the interval mapping, based on maximum likelihood methods described by Lander and Botstein, Genetics 121:185-199 (1989 ) and implemented in the software package MAPMAKER/QTL ( Lincoln and Lander, Mapping Genes Controlling Quantitative Traits Using MAPMAKER/QTL, Whitehead Institute for Biomedical Research, Massachusetts, (1990 ). Additional software includes Qgene, Version 2.23 (1996), Department of Plant Breeding and Biometry, 266 Emerson Hall, Cornell University, Ithaca, NY). Use of Qgene software is a particularly preferred approach.
  • a maximum likelihood estimate (MLE) for the presence of a marker is calculated, together with an MLE assuming no QTL effect, to avoid false positives.
  • the LOD score essentially indicates how much more likely the data are to have arisen assuming the presence of a QTL than in its absence.
  • the LOD threshold value for avoiding a false positive with a given confidence, say 95%, depends on the number of markers and the length of the genome. Graphs indicating LOD thresholds are set forth in Lander and Botstein, Genetics 121:185-199 (1989 ) and further described by Ar ⁇ s and Moreno-González, Plant Breeding, Hayward et al., (eds.) Chapman & Hall, London, pp. 314-331 (1993 ).
  • the nucleic acid marker exhibits a LOD score of greater than 2.0, more preferably greater than 2.5, even more preferably greater than 3.0 or 4.0 with the trait or phenotype of interest.
  • the trait of interest is altered tocopherol levels or compositions.
  • nucleic acid molecules of the invention may be used as molecular markers. It is also understood that one or more of the protein molecules of the invention may be used as molecular markers.
  • the polymorphism is present and screened for in a mapping population, e.g. a collection of plants capable of being used with markers such as polymorphic markers to map the genetic position of traits.
  • a mapping population e.g. a collection of plants capable of being used with markers such as polymorphic markers to map the genetic position of traits.
  • the choice of an appropriate mapping population often depends on the type of marker systems employed (Tanksley et al., J.P. Gustafson and R. Appels (eds.). Plenum Press, New York, pp. 157-173 (1988)). Consideration must be given to the source of parents (adapted vs. exotic) used in the mapping population. Chromosome pairing and recombination rates can be severely disturbed (suppressed) in wide crosses (adapted x exotic) and generally yield greatly reduced linkage distances. Wide crosses will usually provide segregating populations with a relatively large number of polymorphisms when compared to progeny in a narrow cross (adapted x adapted).
  • An F 2 population is the first generation of selfing (self-pollinating) after the hybrid seed is produced.
  • a single F 1 plant is selfed to generate a population segregating for all the genes in Mendelian (1:2:1) pattern.
  • Maximum genetic information is obtained from a completely classified F 2 population using a codominant marker system (Mather, Measurement of Linkage in Heredity: Methuen and Co., (1938)).
  • progeny tests e.g. , F 3 , BCF 2
  • F 3 , BCF 2 progeny tests
  • Progeny testing of F 2 individuals is often used in map construction where phenotypes do not consistently reflect genotype (e.g. disease resistance) or where trait expression is controlled by a QTL.
  • Segregation data from progeny test populations e.g. F 3 or BCF 2
  • Marker-assisted selection can then be applied to cross progeny based on marker-trait map associations (F 2 , F 3 ), where linkage groups have not been completely disassociated by recombination events (i.e. , maximum disequilibrium).
  • Recombinant inbred lines (genetically related lines; usually >F 5 , developed from continuously selfing F 2 lines towards homozygosity) can be used as mapping populations. Information obtained from dominant markers can be maximized by using RILs because all loci are homozygous or nearly so. Under conditions of tight linkage ( i.e. , about ⁇ 10% recombination), dominant and co-dominant markers evaluated in RIL populations provide more information per individual than either marker type in backcross populations ( Reiter. Proc. Natl. Acad. Sci. (U.S.A.) 89:1477-1481 (1992 )). However, as the distance between markers becomes larger (i.e. , loci become more independent), the information in RIL populations decreases dramatically when compared to codominant markers.
  • RIL Recombinant inbred lines
  • Backcross populations (e.g. , generated from a cross between a successful variety (recurrent parent) and another variety (donor parent) carrying a trait not present in the former) can be utilized as a mapping population.
  • a series of backcrosses to the recurrent parent can be made to recover most of its desirable traits.
  • a population is created consisting of individuals nearly like the recurrent parent but each individual carries varying amounts or a mosaic of genomic regions from the donor parent.
  • Backcross populations can be useful for mapping dominant markers if all loci in the recurrent parent are homozygous and the donor and recurrent parent have contrasting polymorphic marker alleles ( Reiter et al., Proc. Natl. Acad. Sci.
  • Backcross populations are more informative (at low marker saturation) when compared to RILs as the distance between linked loci increases in RIL populations ( i.e. about 15% recombination). Increased recombination can be beneficial for resolution of tight linkages, but may be undesirable in the construction of maps with low marker saturation.
  • NIL Near-isogenic lines
  • BSA Bulk segregant analysis
  • the nucleic acid molecule of the present invention is used to determine the level (i.e. , the concentration of mRNA in a sample, etc .) in a plant (preferably canola, maize, Brassica campestris. Brassica napus, soybean, crambe, mustard, castor bean, peanut, sesame, cottonseed, linseed, safflower, oil palm, flax or sunflower) or pattern ( i.e. , the kinetics of expression, rate of decomposition, stability profile, etc .) of the expression of a protein encoded by the nucleic acid molecule of the present invention (collectively, the "Expression Response" of a cell or tissue).
  • a plant preferably canola, maize, Brassica campestris. Brassica napus, soybean, crambe, mustard, castor bean, peanut, sesame, cottonseed, linseed, safflower, oil palm, flax or sunflower
  • pattern i.e. , the
  • the Expression Response manifested by a cell or tissue is said to be "altered” if it differs from the Expression Response of cells or tissues of plants not exhibiting the phenotype.
  • the Expression Response manifested by the cell or tissue of the plant exhibiting the phenotype is compared with that of a similar cell or tissue sample of a plant not exhibiting the phenotype.
  • the phenotype of the organism is any of one or more characteristics of an organism (e.g. disease resistance, pest tolerance, environmental tolerance such as tolerance to abiotic stress, male sterility, quality improvement, yield, etc .).
  • a change in genotype or phenotype may be transient or permanent.
  • a tissue sample is any sample that comprises more than one cell.
  • a tissue sample comprises cells that share a common characteristic (e.g. derived from root, seed, flower, leaf, stem, pollen, etc.).
  • an evaluation can be conducted to determine whether a particular RNA molecule is present.
  • the nucleic acid molecule of the present invention is utilized to detect the presence or quantity of the mRNA species. Such molecules are then incubated with cell or tissue extracts of a plant under conditions sufficient to permit nucleic acid hybridization. The detection of double-stranded probe-mRNA hybrid molecules is indicative of the presence of the mRNA; the amount of such hybrid formed is proportional to the amount of mRNA. Thus, such probes may be used to ascertain the level and extent of the mRNA production in a plant's cells or tissues. Such nucleic acid hybridization may be conducted under quantitative conditions (thereby providing a numerical value of the amount of the mRNA present). Alternatively, the assay may be conducted as a qualitative assay that indicates either that the mRNA is present, or that its level exceeds a user set, predefined value.
  • a number of methods can be used to compare the expression response between two or more samples of cells or tissues. These methods include hybridization assays, such as northerns, RNAse protection assays, and in situ hybridization. Alternatively, the methods include PCR-type assays. In a preferred method, the expression response is compared by hybridizing nucleic acids from the two or more samples to an array of nucleic acids. The array contains a plurality of suspected sequences known or suspected of being present in the cells or tissue of the samples.
  • in situ hybridization over more conventional techniques for the detection of nucleic acids is that it allows an investigator to determine the precise spatial population ( Angerer et al., Dev. Biol. 101:477-484 (1984 ); Angerer et al., Dev. Biol. 112:157-166 (1985 ); Dixon et al., EMABO J. 10:1317-1324 (1991 )).
  • In situ hybridization may be used to measure the steady-state level ofRNA accumulation ( Hardin et al., J. Mol. Biol. 202:417-43 (1989 )).
  • a number of protocols have been devised for in situ hybridization, each with tissue preparation, hybridization, and washing conditions ( Meyerowitz, Plant Mol. Biol. Rep.
  • In situ hybridization also allows for the localization of proteins within a tissue or cell ( Wilkinson, In Situ Hybridisation, Oxford University Press, Oxford (1992 ); Langdale, In Situ Hybridization In: The Maize Handbook, Freeling and Walbot (eds.), pp. 165-179, Springer-Verlag, New York (1994 )). It is understood that one or more of the molecules of the invention, preferably the nucleic acid molecule of the invention, or one or more of the antibodies of the invention, may be utilized to detect the level or pattern of a protein or mRNA thereof by in situ hybridization.
  • Fluorescent in situ hybridization allows the localization of a particular DNA sequence along a chromosome, which is useful, among other uses, for gene mapping, following chromosomes in hybrid lines, or detecting chromosomes with translocations, transversions or deletions.
  • In situ hybridization has been used to identify chromosomes in several plant species ( Griffor et al., Plant Mol. Biol. 17:101-109 (1991 ); Gustafson et al., Proc. Natl. Acad. Sci.
  • nucleic acid molecule of the invention may be used as probe or marker to localize sequences along a chromosome.
  • tissue printing Another method to localize the expression of a molecule is tissue printing.
  • Tissue printing provides a way to screen, at the same time and on the same membrane, many tissue sections from different plants or at different developmental stages ( Yomo and Taylor, Planta 112:35-43 (1973 ); Harris and Chrispeels, Plant Physiol. 56:292-299 (1975 ); Cassab and Varner, J. Cell. Biol.
  • a published partial sequence of a putative AANT1 ( Saint-Guily et al., Plant Physiol. 100(2), 1069-1071 (1992 )) is used to search an EST database, and three full-length clones are identified: LIB3176-057-P1-K1-B12, LIB3234-001-P1-K1-G3, and LIB3176-083-P1-K1-F2.
  • An ANT1 coding region is PCR-amplified using the forward primer AANT1F 5'- GGA TCC GCG GCC GC A CCA TGG TTG ATC AAG TTC AGC A (SEQ ID NO: 3) and the reverse primer AANT1R 5'-GAG CTC CTG CAG G AA GCT TIT AGG CAC CTC CTG ATC CGT-3' (SEQ ID NO: 4).
  • the AANT1F primer contains a Not I site (underlined) upstream of the start codon (italics) while the AANT1R primer contains an Sse 83S7I site (underlined) downstream of the stop codon (italics).
  • a PCR product is generated and cloned into pCR2.1, and the insert is verified by sequencing both strands.
  • the Not I/ Sse 8387I fragment from example 1 is inserted into the Not I/ Sse 8387I sites of the napin expression cassettes in pCGN9979, which is shown in Figure 2 , and pCGN11307, which is shown in Figure 3 .
  • pCGN9979 is designed for use in Arabidopsis
  • pCGN11307 is designed for use in Brassica.
  • Both pCGN9979 and pCGN11307 contain a seed specific expression cassette using the Brassica seed storage protein (napin) gene promoter ( Krid1 et al., Seed Sci. Res. 1:209-219 (1991 )).
  • the resulting plasmids are pCGN11301 (for pCGN9979) and pMON66454 (for pCGN11307), which are shown in Figures 4 and 5 , respectively.
  • Kordopsis plants are grown for transformation with pCGN11301.
  • Kord pots are filled with Metro Mix 200 soil. Pots are placed into Kord 1040 green flats with holes, which are placed into Kord 1040 white flats without holes.
  • the flats are filled with 1" of reverse osmosis water and a humidity dome is placed over each of the flats. The pots are allowed to soak up water for several hours to overnight until the soil is completely moistened. Eight inch fiberglass screens are placed over the soil and secured and seeds are sown.
  • the flats are placed in a chamber at 22° C for a 16/8 photoperiod at 55% relative humidity and 234 ⁇ Mol/s/m 2 . After germination the domes are raised slightly and then removed after three days.
  • the plants are bottom watered as needed with Plantex 15-15-18 at 50 PPM N.
  • the plants are thinned down to 7 plants per pot 3 weeks after seeding.
  • the primary bolts at the base of the plant are pruned to promote secondary growth.
  • the plants are bottom watered to saturate the soil prior to transformation. Plants are ready for transformation when secondary bolts are emerging (for vacuum infiltration transformation) or when bolts are 2-10 cm (for dipping method of transformation).
  • LB 75 mg/l spectinomycin, 50 mg/l kanamycin, and 25 mg/l chloramphenicol
  • the 6 ml culture is then added to 300 ml of the same medium in a 1 liter Erlenmeyer flask and the culture is incubated at the same conditions for about 18 hours.
  • the pots containing the plants to be transformed are immersed in water for about 30 minutes prior to dipping.
  • the Agrobacterium culture is centrifuged at 5000 x g for 12 minutes at 15°C, and the pellet is resuspended in 300 ml of 5% sucrose solution in water. Fifty ml aliquots of this suspension are placed into 200 ml of 5% sucrose solution, to which 125 ⁇ l of Silwet L-77 (Lehle Seeds) has been added. This mixture is poured into a plastic dish of about 14 cm diameter, with a capacity of about 850 ml.
  • the above-ground portion of the plants are dipped in the Agrobacterium solution for 2-5 seconds, with gentle agitation. Excess Agrobacterium solution is blotted with paper towels. The dipped plants are placed on their sides with the bolts on top of paper towels, and then are placed in a flat and covered with a plastic dome. The cover is kept in place for 16 to 25 hours to maintain high humidity.
  • the pots are not watered for about 5 days following dipping. After 5 days, the pots are provided with water and the plants are grown under normal conditions. Watering is stopped when the seeds become mature (in about 4 weeks) and the dry seed is harvested.
  • Selection involves sprinkling the transformed T 1 seeds directly onto the soil and then vernalizing them at 4°C in 8 hours light, 16 hours dark for 4-7 days. The seeds are then transferred to 22°C in 16 hours light and sprayed with a 1:200 dilution of Finale (Basta) at 7 days and again at 14 days.
  • Agrobacterium strains ABI, EHA105, and EHA101 can be used for canola transformation.
  • the canola cultivar Ebony can be used.
  • culture room shelves are lit by two Cool White HO, and one Gro-Lux HO (or equivalent), fluorescent bulbs per shelf.
  • the temperature of culture rooms is 22°C.
  • the seeds are surface-sterilized in a wire mesh tea strainer, for 2 minutes in 95% ethanol or 70% isopropanol, followed by 30 minutes in 20% bleach solution (1% NaOCl) with one drop of Tween 20 per 100 ml. The seeds are then rinsed 4 times with sterile reverse osmosis water. Sterilized seed is plated on modified 1/10X MS medium (1/10X MS minimal organics medium (Gibco 510-1118; final sucrose 0.3%), pyridoxine 50ug/l, nicotinic acid 50ug/l, glycine 200 ug/l, Phytagar (Gibco) 6 g/l, pH 5.8; 20 boxes per liter) at 25 seeds per Magenta box. The seed is kept at 24°C for 16 hours light/day and 15-35 uE/m2/sec under shade. Complete darkness may be used during the last 24 hours to increase etiolation.
  • modified 1/10X MS medium (1/10X MS minimal organics
  • Brassica napus feeder plates are prepared 1 day before explant cutting.
  • hypocotyl segments (2-4mm) are excised from etiolated one-week-old seedlings (4 to 14 days old). Segments are cut in a petri dish with several ml of water to prevent dehydration. Explants are placed onto a filter paper (Whatman 1001-082) and laid over the tobacco feeder cells on MS 0/1/0 cocultivation medium (MS salts (Gibco 500-1117), myo-inositol 100 mg/l, thiamine-HCl 1.3 mg/l, KH 2 PO 4 200 mg/l, 2,4-D1 mg/l, sucrose 3%, Phytagar 6 g/l, pH 5.8; 30 plates per liter) at 50 explants per plate (100 x 15 mm) and the plates are stacked in a sealed plastic sleeve and incubated for 18-24 hours at 24°C in indirect continuous light.
  • MS salts Gibco 500-1117
  • myo-inositol 100 mg/l thiamine-HCl 1.3 mg
  • a single colony of Agrobacterium containing pMON66454 is grown overnight in 5 ml MG/L broth (Mannitol 5 g/l, L-glutamic acid 1 g/l, KH2PO4 250 mg/l, NaCl 100 mg/l, MgSO4.7h20 100 mg/l, biotin 1 ug/l, tryptone 5 g/l, yeast extract 2.5 g/l, pH 7.0) in a large culture tube, at 30°C, in the dark, at 200 rpm.
  • the Agrobacterium suspension OD is measured at 550 nm on a spectrophotometer.
  • the overnight culture is diluted with MG/L broth to 0.05 OD (equivalent to about 1x10 8 bacteria/ml).
  • the preincubated explants are inoculated for 10 minutes in 1x10 8 bacteria/ml; 200-400 explants per 10 ml inoculum. Inoculated explants are drained well, returned to the feeder plates, and cocultivated with Agrobacterium for 2 days. Plates are stacked in a sealed plastic sleeve and incubated at 24°C in indirect continuous light.
  • Cocultivated explants are transferred to B5 0/1/0 callus induction medium (B5 salts (Gibco 85-5068), B5 vitamins (1 mg/l nicotinic acid, 1 mg/l pyridoxine-HCl, 10 mg/l thiamine-HCl), 100 mg/l inositol, 1 mg/l 2,4-D, sucrose 3%, 500 mg/l carbenicillin, 50 mg/l Timentin, 6 g/l Phytagar, pH5.8, 30 plates per liter) supplemented with 500 mg/l carbenicillin and 50 mg/l Timentin to inhibit Agrobacterium. There are about 40-50 explants per plate (100 x 15 mm). The plates are sealed with Parafilm.
  • Explants remain on B5 0/1/0 for 7 days (to achieve a total of about 10 days exposure to 2,4-D) at 24°C in continuous light at 85 uE/m2/s.
  • Explants are transferred to B5BZ shoot regeneration medium (B5 salts (Gibco 85-5068), B5 vitamins (1 mg/l nicotinic acid, 1 mg/l pyridoxine-HCl, 10 mg/l thiamine-HCl), 100 mg/l inositol, BAP 3 mg/l (Sigma B3408), zeatin 1 mg/l (Sigma Z0164), sucrose 1%, Phytagar 6 mg/l, pH 5.8; 25 plates per liter, silver nitrate (3 mg/l) is included during the first two weeks on B5BZ medium) containing 500 mg/l carbenicillin with 45 mg/l glyphosate.
  • B5BZ shoot regeneration medium B5 salts (Gibco 85-5068)
  • B5 vitamins (1 mg/
  • the plates (100 x 25 mm) are sealed with Parafilm and incubated at 24°C in continuous light at 85 uE/m2/s. Explants are transferred to fresh medium every second week and plating density is reduced to 20-25 explants after two weeks on B5BZ. Shoots regenerate from kanamycin-selected calli 5 to 9 weeks after culture initiation.
  • B5 salts (Gibco 85-5068) B5 vitamins (1 mg/l nicotinic acid, 1 mg/l pyridoxine-HCl, 10 mg/l thiamine-HCl), 100 mg/l inositol, sucrose 1%, Phytagar 6 g/l, pH 5.8; 25 plates per liter) containing 300 mg/l carbenicillin and 45 mg/l glyphosate. Multiple shoots from a single callus are kept together as one event with 9 events per plate. The plates are sealed with parafilm and shoots remain on maturation medium for 7-14 days at 24°C for 16 hours light/day at 85 uE/m2/s.
  • B5 root induction medium B5 salts (Gibco 85-5068), B5 vitamins (1 mg/l nicotinic acid, 1 mg/l pyridoxine-HCl, 10 mg/l thiamine-HCl), 100 mg/l inositol, IBA2 mg/l, sucrose 1%, Phytagar 6 g/l, pH 5.8; 20 boxes per liter) containing 45 mg/l glyphosate with 5 events per Magenta box at 24°C for 16 hours light/8 hours dark at 85 uE/m2/s. Roots develop over a period of 4-6 weeks and stems are re-cut to encourage rooting, as needed.
  • FIG. 1 shows the total seed tocopherol level (nanograms per milligram) for 20 plant lines. Tocopherol levels are determined by adding 10 to 15 mg of Arabidopsis seed into a 2 mL microtube. A mass of 1g of 0.5mm microbeads (Biospecifics Technologies Corp., Lynbrook, NY) and 500 ⁇ l of 1% pyrogallol (Sigma Chem, St. Louis, MO) in ethanol containing 5 ⁇ g/mL tocol, are added to the tube.
  • 0.5mm microbeads Biospecifics Technologies Corp., Lynbrook, NY
  • 500 ⁇ l of 1% pyrogallol Sigma Chem, St. Louis, MO
  • the sample is shaken twice for 45 seconds in a FastPrep (Bio101/Savant) at a speed of 6.5.
  • the extract is filtered (Gelman PTFE acrodisc 0.2 ⁇ m, 13 mm syringe filters, Pall Gelman Laboratory Inc, Ann Arbor, MI) into an autosampler tube.
  • HPLC is performed on a Zorbax silica HPLC column, 4.6 mm x 250 mm (5 ⁇ m) with a fluorescent detection using a Hewlett Packard HPLC (Agilent Technologies, Palo Alto CA). Sample excitation is performed at 290 nm, and emission is monitored at 336 nm.
  • Tocopherols are separated with a hexane methyl-t-butyl ether gradient using an injection volume of 20 ⁇ l, a flow rate of 1.5 ml/min, and a run time of 12 min (40°C). Tocopherol concentration and composition is calculated based on standard curves for ⁇ , ⁇ , ⁇ , and ⁇ -tocopherol using Chemstation software (Agilent Technologies, Palo Alto CA).
  • Canola, Brassica napus and soybean plants are transformed with a variety of DNA constructs using a particle bombardment approach essentially as set forth in Christou, In Particle Bombardment for the Genetic Engineering of Plants, Biotechnology Intelligence Unit, Academic Press, San Diego, California (1996 ) or using Agrobacterium mediated transformation.
  • Two sets of DNA constructs are produced. The first set of constructs are "single gene constructs.” Each of the following genes is inserted into a separate plant DNA construct under the control of a napin promoter ( Kridl et al., Seed Sci. Res.
  • the products of the genes can be targeted to the plastid by an encoded plastid target peptide such as CTP1 ( Keegstra, Cell 56(2):247-53 (1989 ); Nawrath, et al., Proc. Natl. Acad. Sci. U.S.A. 91:12760-12764 (1994 )): an E. herbicola tyrA gene ( Xia et al., J. Gen. Microbiol.
  • CTP1 Keegstra, Cell 56(2):247-53 (1989 ); Nawrath, et al., Proc. Natl. Acad. Sci. U.S.A. 91:12760-12764 (1994 )
  • E. herbicola tyrA gene Xia et al., J. Gen. Microbiol.
  • a AANT1 gene Saint Guily, et al., Plant Physiol, 100(2):1069-1071 (1992 )
  • a GMT gene WO 00/32757 , WO 00/10350
  • a slr1737 gene in Cyanobase (www.kazusa.or.jp/cyanobase)
  • an MT1 gene The sequence of the Synechocystis MT1 (NCBI General Identifier Number1653572) was used in a blast search against ESTs of Anabaena sp.
  • strain PCC 7120 (Kaneko 2001) and a sequences with substantial homology to the Synechocystis MT1 was found in a blast search against ESTs of Anabaena sp. strain PCC 7120 ( Kaneko et al., DNA Research 8(5): 205-213 (2001 )), a TMT2 gene (as disclosed in US 2003/150015 ), and an antisense construct for homogentisic acid dioxygenase ( Sato et al., J. DNA Res. 7 (1):31-63 (2000 )).
  • Each construct is transformed into at least one canola, Brassica napus, and soybean plant. Plants expressing each of these genes are selected to participate in additional crosses.
  • the tocopherol composition and level in each plant is also analyzed using the method set forth in example 5.
  • Crosses are carried out for each species to generate transgenic plants having one or more of the following combinations of introduced genes: tyrA, slr1736, ATP72, dxs, dxr, GGPPS, HPPD, GMT.
  • the tocopherol composition and level in each plant generated by the crosses is also analyzed using the method set forth in example 5. Progeny of the transformants from these constructs will be crossed with each other to stack the additional genes to reach the desired level of tocopherol.
  • a second set of DNA constructs is generated and referred to as the "multiple gene constructs".
  • the multiple gene constructs contain multiple genes each under the control of a napin promoter ( Kridl et al., Seed Sci. Res. 1:209:219 (1991 )) and the products of each of the genes are targeted to the plastid by an encoded plastid target peptide.
  • the multiple gene construct can have two or more of the following genes: tyrA, slr1736, ATPT2, dxs, dxr, GGPPS, HPPD, GMT, AANT1, slr 1737, MT1, TMT2, and an antisense construct for homogentisic acid dioxygenase.
  • Each construct is then transformed into at least one canola, Brassica napus and soybean plant.
  • the tocopherol composition and level in each plant is also analyzed using the method set forth in example 5. Progeny of the transformants from these constructs will be crossed with each other to stack the additional genes to reach the desired level of tocopherol.
  • Example 7 Transformation of Canola with pMON66454 and measurement of total tocopherol composition.
  • Canola plants are transformed using Agrobacterium as in Example 4 with the vector pMON66454 (described in Example 2) which contains a coding region for AANT1. Plants are grown, and seeds are harvested and tested for tocopherol levels essentially as described in Example 5 for Arabidopsis seeds. Table 1 shows the total seed tocopherol level for control plants and several plants generated from separate transformation events (given in nanograms per milligram of seed). The average tocopherol levels expressed in control seed is 325 +/- 36 ng/mg of seed, and the average tocopherol levels for seed from R1 plants bearing the vector pMON66454 is 338 +/- 36 ng/mg of seed.
  • Example 8 Transformed Plants with AANT1 and other tocopherol biosynthesis genes.
  • Canola, Brassica napus, Arabidopsis, and soybean plants are transformed with a variety of DNA constructs using a particle bombardment approach essentially as set forth in Christou, In Particle Bombardment for the Genetic Engineering of Plants, Biotechnology Intelligence Unit, Academic Press, San Diego, California (1996 ) or using Agrobacterium mediated transformation.
  • Two sets of DNA constructs are produced. The first set of constructs are "single gene constructs". Each of the following genes is inserted into a separate plant DNA construct under the control of a napin promoter ( Kridl et al., Seed Sci. Res.
  • an slr1736 gene in Cyanobase on the world wide web at: kazusa.or.jp/cyanobase
  • a plant ATPT2 gene Smith et al., Plant J. 11: 83-92 (1997 )
  • a dxs gene Lois et al., Proc. Natl. Acad. Sci. U.S.A. 95 (5):2105-2110 (1998 )
  • a dxr gene Takahashi et al. Proc. Natl. Acad. Sci. U.S.A.
  • strain PCC 7120 Kaneko et al., DNA Research 8(5): 205-213 (2001 )
  • TMT2 gene as disclosed in US 2003/150015
  • GMT gene as disclosed in US 2003/154513 and WO 00/32757 , WO 00/10380
  • an slr1737 gene in Cyanobase (on the world wide web at kazusa.or.jp/cyanobase), and an antisense construct for homogentisic acid dioxygenase ( Sato et al., J. DNA Res. 7 (1):31-63 (2000 ))).
  • Each construct is transformed into at least one canola, Brassica napus, Arabidposis and soybean plant.
  • Plants expressing each of these genes are selected to participate in additional crosses.
  • the tocopherol composition and level in each plant is also analyzed using the method set forth in example 5.
  • Crosses are carried out for each species to generate transgenic plants having one or more of the following combination of introduced genes: tyrA, slr1736, ATPT2, dxs, dxr, GGH, GGPPS, HPPD, MT1, TMT2, GMT, AANT1, slr 1737, and an antisense construct for homogentisic acid dioxygenase.
  • the tocopherol composition and level in each plant generated by the crosses is also analyzed using the method set forth in example 5. Progeny of the transformants from these constructs are crossed with each other to stack the additional genes to reach the desired level of tocopherol.
  • a second set of DNA constructs is generated and referred to as the "multiple gene constructs.”
  • the multiple gene constructs contain multiple genes each under the control of a napin promoter ( Kridl et al., Seed Sci. Res. 1:209:219 (1991 )) and the products of , each of the genes are targeted to the plastid by an encoded plastid target peptide.
  • the multiple gene construct can have two or more of the following genes: tyrA, slr1736, ATPT2, dxs, dxr, GGH, GGPPS, HPPD, MT1, TMT2, GMT, AANT1, slr 1737, and an antisense construct for homogentisic acid dioxygenase.
  • Each construct is then transformed into at least one canola, Brassica napus, Arabidopsis, and soybean plant.
  • the tocopherol composition and level in each plant is also analyzed using the method set forth in example 5. Progeny of the transformants from these constructs are crossed with each other to stack the additional genes to reach the desired level of tocopherol.
  • Expression constructs pCGN10822, pMON36528, pMON69907 and pMON69909, shown in figures 10-13 respectively, are prepared. Arabidopsis plants are transformed with the indicated vectors using the transformation techniques described in Example 8. Transformants are isolated and grown into individual lines by self pollination and seed from each line collected. The total tocopherol and tocotrienol composition of the seeds from each line are analyzed using the method set forth in Example 5. Figure 10 shows total tocopherol and tocotrienol levels for plant lines harboring the described contructs or a control. An analysis of T2 seeds from plant lines derived by transformation with the vector pMON69909 relative to wild type is shown in figure 11 .
  • Plant lines transformed with pMON69909 demonstrate a substantial increase in total tocopherols and total tocotrienols, with the largest increases in delta tocopherol, alpha tocotrieneol, delta tocotrienol, and gamma tocotrienol. Some seed from plants harboring the vector pMON69909 show a dark coloration as the result of homogentisic acid accumulation, which is confirmed by LC/MS analysis (see Figures 12 and 13 ).
  • Heterologous expression of tyrA in seeds of transgenic Arabidopsis plants produces a 1.6-fold increase in seed tocopherol levels as compared to control lines.
  • Another key enzyme essential for tocopherol biosynthesis is HPT, which is involved in the condensation of phytyl pyrophosphate (PPP) and homogentisate (HGA) to produced 2-methyl-6-phytylplastoquinol (2M6PPQ), a precursor for synthesis of four different isoforms of tocopherols.
  • HPT Arabidopsis (ATPT2) and the HPT Synechocystis (slr1736) independently in seeds of transgenic A . thaliana results in a 1.6-fold increase in seed tocopherols.
  • a putative adenylate transporter from A. thaliana (AANT1) expressed as a single gene is shown to increase seed tocopherol levels to 1.4-fold in A. thaliana. To test whether a combination of these genes would result in synergistic effect on tocopherol biosynthesis, various combinations are tested in A. thaliana.
  • HPPD expressed as a single gene in A. thaliana result in a barely detectable increase of tocopherol levels.
  • the combination of HPPD with APT2 does not result in a further increase of tocopherol levels as compared to lines harboring ATPT2 alone (data not shown).
  • tocopherol and tocotrienol levels double compared to the tyrA, ATPT2 combination.
  • Seeds harboring the triple gene construct pMON69909 appear much darker in color than control seeds.
  • wild-type dicotyledonous plants do not accumulate tocotrienols.
  • the transgenic A is known that wild-type dicotyledonous plants do not accumulate tocotrienols.
  • the transgenic A is known that wild-type dicotyledonous plants do not accumulate tocotrienols.
  • the transgenic A is known that wild-type dicotyledonous plants do not accumulate tocotrienols.
  • the transgenic A is known that wild-type dicotyledonous plants do not
  • thaliana seeds harboring all four constructs accumulates substantial levels of tocotrienols (confirmed by HPLC, and for selected samples by LC-MS, (See Figures 12 / 13 , 14 , and 15 ).
  • the tocopherol and tocotrienol content of seeds harboring the triple gene expression construct, pMON69909 consist of 60% tocotrienols and 40% tocopherols.
  • the HPT When the availability of endogenous HGA is elevated by overexpression of the HGA biosynthetic enzymes ( tyrA & HPPD ) along with HPT, the HPT would utilize geranylgeranyl pyrophosphate ( GGPP ) and HGA to produce tocotrienols instead of tocopherols under conditions limited by the availability of endogenous level of geranylgeranyl reductase ( GGH ).
  • GGH functions on hydrogenating the GGPP to PPP, a substrate for HPT in tocopherol synthesis.
  • increased tocotrienols accumulation seen in the constructs tested can be overcome by overexpression of GGH in combination with tyr A, HPPD , and HPT .
  • Example 10 Transformed plants with AANT1 and other tocopherol biosynthesis genes.
  • Plants are transformed with the DNA constructs shown a tables 2 and 3. below, employing the techniques described in Example 8.
  • the constructs contain one or more genes under the control of a napin promoter (kind et al., Seed Sci. Res. 1:209:219 (1991 )), the 7S 'promoter ( Chen et al., PNAS 83 (22):8560-8564 (1998 )) or the Arcelin 5 (Arc5) promoter ( Goossens et al., Plant Physiol. 120:1095 1104 (1999 ), and as disclosed in US 2005/0241018 and US 2003/0046727 ).
  • a napin promoter Kind et al., Seed Sci. Res. 1:209:219 (1991 )
  • the 7S 'promoter Chen et al., PNAS 83 (22):8560-8564 (1998 )
  • Arcelin 5 (Arc5) promoter Goossens et al., Plant Physiol. 120:10
  • the products of the genes can be targeted to the plastid by an encoded plastid target peptide such as CTP1 ( Keegstra, Cell 56(2):247-53 (1989 ); Nawrath, et al., Proc. Natl. Acad. Sci. U.S.A. 91:12760-12764 (1994 )) or CTP2.
  • CTP1 Enegstra, Cell 56(2):247-53 (1989 ); Nawrath, et al., Proc. Natl. Acad. Sci. U.S.A. 91:12760-12764 (1994 )
  • CTP2 CTP1
  • E. herbicola tyrA gene Xia et al., J. Gen. Microbiol.
  • an AANT1 gene Saint Guily, et al., Plant Physiol., 100(2):1069-1071 (1992 )
  • an MT1 gene (as above for Example 8)
  • a TMT2 gene (as above for example 8)
  • a GMT gene (as above for example 8, and WO 00/32757 , WO 00/10380 )
  • an slr1737 gene (in Cyanobase on the world wide web at kazusa.org jp/cyanobase), and an antisense construct for homogentisic acid dioxygenase (denoted HGD AS )( Sato et al., J. DNA Res. 7 (1):31-63 (2000 )).
  • Each construct is transformed into at least one canola, Brassica napus, Arabidposis, and soybean plant.
  • the tocopherol composition and level in each plant is also analyzed using the method set forth in example 5.
  • Examples of transformed plants with tyrA and other tocopherol biosynthesis genes include Arabidopsis plants transformed with the constructs set forth in Table 2 and soy plants transformed with the constructs in Table 3.
  • Plants with desired characteristics are subject to further crosses to generate transgenic plants having one or more of the following combination of introduced genes: tyrA, slr1736, ATPT2, dxs, dxr, GGH, GGPPS, HPPD, MT1, TMT2, GMT, AANT1, slr 1737, and an antisense construct for homogentisic acid dioxygenase.
  • the plants are crossed to stack multiple copies of one or more of the aforementioned genes in a transgenic plant.
  • TABLE 2 The following gene combinations are prepared and tested in Arabidopsis thaliana 1 pNapin::CTP1::TyrA E.
  • thaliana ::Napin3'/pNapin::ATPT2 A thaliana ::Napin 3' 4 pNapin::CTP1::TyrA E . herbicola ::Napin 3'/pNapin::CTP1::DXS E . coli ::Napin3'/pNapin::ATPT2 A .
  • tyrA prephenate dehydrogenase
  • the enzymes combined with tyrA include ATPT2, p- hydroxyphenylpyruvate dioxygenase (HPPD Arabidopsis ), and geranylgeranylpyrophosphate synthase (GGPPS Ar abidopsis ) from Arabidopsis thaliana.
  • tyrA is also tested in combination with ATPT2 and an adenylate transporter (AANT1 Arabidopsis ) from Arabidopsis thaliana.
  • a double gene vector harboring seed specific tyrA and ATPT2 expression cassettes is performed as follows. Purified plasmid DNA of pMON36520 ( Figure 18 ) is subjected to a partial KpnI digest and ligated with a 4.2 kbp gel purified Kpn I-fragment isolated from pMON43853 ( Figure 19 ). The 4.2 kb insert from pMON43853 contains the PPT gene expression cassette (pNapin::ATPT2::Napin 3'). The resulting plant binary vector pMON69907 ( Figure 8 ) is used for transformation of Arabidopsis thaliana to test the combinatorial effect of seed specific expression of tyrA and ATPT2.
  • HPPD Arabidopsis is expressed in addition to tyrA, and ATPT2 in Arabidopsis thaliana seed. This is achieved by adding a seed specific expression cassette for HPPD Arabidopsis to pMON69907 resulting in the formation of pMON69909.
  • the binary vector pMON69909 is constructed by partially digesting pMON69907 with KpnI.
  • the single KpnI-cut pMON69907 is gel purified and ligated with a 4.6 kb KpnI/KpnI insert isolated from pMON36525 ( Figure 20 ).
  • the 4.6 kb KpnI/KpnI insert from pMON36525 contains the HPPD gene expression cassette, pNapin::CTP2::HPPD Arabidopsis ::Napin 3' to direct seed specific plastid targeted expression of HPPD.
  • the CTP2 is a chloroplast-target signal from the Arabidopsis 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene.
  • EPSPS Arabidopsis 5-enolpyruvylshikimate-3-phosphate synthase
  • the binary vector pMON69915 ( Figure 16 ) is constructed to test the effect of three gene combinations, tyrA, ATPT2, and GGPPS Arabidopsis on seed tocopherol production.
  • Vector pMON69907 is digested partially with KpnI.
  • Single KpnI-cut pMON69907 is gel purified and ligated with a gel purified 4.3 kb, KpnI/KpnI fragment from pMON43861 ( Figure 21 ) to create pMON69915.
  • the KpnI fragment from pMON43861 contains the gene expression cassette for the Arabidopsis geranylgeranyldiphosphate synthase from Arabidopsis thaliana (pNapin::GGPPS Arabidopsis ::Napin 3').
  • the GGPPS cDNA is identified as an EST clone by searching an EST database with sequence information available in the literature ( Okada et al., Plant Physiol. 122:1045-1056 (2000 )).
  • the EST clone is digested with NcoI and blunt-ended by filling the 5' overhang with the Klenow-fragment.
  • the clone is digested with BamHI and to excise the cDNA fragment.
  • the gel purified BamHI/blunt cDNA fragment is ligated with BglII/SalI digested and (SalI blunt-ended) vector pCGN7770 ( Figure 22 ) to create pMON43861.
  • the plant binary vector pMON69919 ( Figure 17 ) is constructed to test combined expression of tyrA, ATPT2, and AANT1 Arabidopsis on seed tocopherol levels.
  • pMON69907 is partially digested with KpnI.
  • Single KpnI-cut pMON69907 is gel purified, and ligated with a 4.2 kb gel-purified KpnI/KpnI fragment from pMON69911 ( Figure 23 ).
  • the 4.2 kb fragment contains a seed specific expression cassette for the Arabidopsis adenylate transporter AANT1 (pNapin::AANT1 Arabidopsis ::napin 3').
  • pMON69911 is generated by excising the AANT1 fragment from pCGN11301 ( Figure 24 ) with SalI and PstI (the PstI site is blunted by removing 3' overhang with Klenow) and then ligated to SalI/XhoI digested (XhoI blunt-ended) pCGN7770.
  • AANT1 Using the published partial sequence of AANT1 ( Saint-Guily et al., Plant Physiol. 100(2):1069-1071 (1992 )) several full-length clones are identified in EST databases.
  • the AANT1 coding region is PCR-amplified using primers, AANT1F 5'-GGATCC GCGGCCGC ACG ATG GTTGATCAAGTTCAGCA (SEQ ID NO: 11) and AANT1R 5'-GAGCT CCTGCAGG AAGCTT TTA GGCACCTCCTGATCCGT-3' (SEQ ID NO: 12).
  • the Not I site (underlined) is placed upstream of the start codon (italics) in primer AANT1F while the Sse8387I site (underlined) is placed downstream of the stop codon (italics) in AANT1R.
  • the PCR products are first cloned into pCR2.1 and the inserts are verified by sequencing of both strands. Subsequently, the Not I/ Sse 8387I fragments are inserted into the Not I/ Sse 8387I sites of the napin expression cassette in pCGN9979 in sense orientation with respect to the napin promoter to generate pCGN11301.
  • the plant expression constructs are used for transformation of Arabidopsis thaliana by Agrobacterium mediated transformation as described in above.

Claims (17)

  1. Molécule d'acide nucléique, où ladite molécule d'acide nucléique code pour la protéine de transport d'adénylate et est choisie parmi
    (a) une molécule d'acide nucléique codant pour le polypeptide ayant la séquence de SEQ ID NO : 2,
    (b) une molécule d'acide nucléique comprenant la séquence d'acide nucléique de SEQ ID NO : 1,
    où ladite molécule d'acide nucléique est liée de manière fonctionnelle à un promoteur.
  2. Molécule d'acide nucléique selon la revendication 1, où ledit promoteur fournit une expression à spécificité tissulaire ou cellulaire, en particulier spécifique du tubercule, du fruit, de la feuille, de la graine, de l'endosperme, de l'ovule, du pollen, de l'aleurone, de la racine ou de la tige.
  3. Molécule d'acide nucléique selon la revendication 1 ou 2, où ledit promoteur est un promoteur spécifique de la graine tel qu'un promoteur de la napine ou un promoteur 7S.
  4. Molécule d'acide nucléique selon la revendication 1, où ledit promoteur est choisi parmi le promoteur de la nopaline synthase, le promoteur de l'octopine synthase, le promoteur 19S ou 35S du virus de la mosaïque du chou-fleur, le promoteur 35S du virus de la mosaïque de la scrofulaire, le promoteur de l'Adh, le promoteur de la saccharose synthase, le promoteur du complexe du gène R, le promoteur de la protéine de liaison de la chlorophylle a/b, le promoteur GS2 de la glutamine synthétase, le promoteur de la fructose-1,6-biphosphatase (FBPase), le promoteur de ST-LS1, le promoteur de la sérine/thréonine kinase, le promoteur de la glucoamylase, le promoteur de la ribulose-1,5-biphosphate carboxylase (RbcS), le promoteur du gène cab, le promoteur de Cab-1 issu du blé, de l'épinard ou du riz, le promoteur de la pyruvate orthophosphate dikinase (PPDK), le promoteur de Lhcb1*2, le promoteur du symport saccharose-H+ SUC2, le promoteur de LhcB, le promoteur de PsbP, un promoteur de patatine de classe I, un promoteur de patatine de classe II, le promoteur de l'ADPGPP du tubercule de pomme de terre, un promoteur de protéine de liaison de la chlorophylle a/b, un promoteur de protéine membranaire du thylakoïde, le promoteur de la saccharose synthase, le promoteur du complexe de la protéine de 22 kd du tubercule, un promoteur d'inhibiteur de protéase du tubercule, le promoteur de l'amidon synthase liée aux granules, le promoteur de la napine, le promoteur de la phaséoline, le promoteur d'inhibiteur de la trypsine du soja, le promoteur de l'ACP, le promoteur de la stéaroyl-ACP désaturase, le promoteur 7S du soja, le promoteur de l'oléosine, le promoteur de la β-conglycinine, un promoteur de la zéine, le promoteur du gène waxy, le promoteur du gène Brittle, le promoteur du gène Shrunken 2, le promoteur de l'enzyme structurante I, le promoteur de l'enzyme structurante II, un promoteur de l'amidon synthase, un promoteur d'enzyme déstructurante, un promoteur d'oléosine, un promoteur de glutéline, un promoteur de gliadine, un promoteur de gluténine, un promoteur de saccharose synthase, le promoteur de l'Osgt-1, un promoteur pour les sous-unités de l'ADP-glucose pyrosynthase, un promoteur de protéine abondante dans l'embryogenèse, un promoteur de l'hordéine, un promoteur de globuline d'embryon, un promoteur de protéine spécifique de l'aleurone, un promoteur de l'arceline 5, le promoteur de la chitinase acide et les sous-domaines spécifiques de la racine du promoteur 35S du CaMV.
  5. Molécule d'acide nucléique qui comprend : (A) une région de promoteur qui fonctionne dans une cellule végétale pour provoquer la production d'une molécule d'ARNm ; (B) une molécule d'acide nucléique structural exogène codant pour une protéine comprenant une séquence d'acides aminés de SEQ ID NO : 2 ; et (C) une séquence non traduite en 3' qui fonctionne dans ladite cellule végétale pour provoquer la terminaison de la transcription et de l'addition de ribonucléotides polyadénylés à une extrémité 3' de la molécule d'ARNm.
  6. Molécule d'acide nucléique comprenant en tant que composants liés de manière fonctionnelle : (A) une région de promoteur qui fonctionne dans une cellule végétale pour provoquer la production d'une molécule d'ARNm ; (B) une séquence d'acide nucléique qui code pour un transporteur d'adénylate ayant la séquence de SEQ ID NO : 2 ; où ladite molécule d'acide nucléique comprend en outre (C) une ou plusieurs cassettes d'expression, dont chacune exprime un membre choisi parmi la HPT de Synechocystis (homogentisate phytyltransférase ; slr1736), la prényltransférase 2 de Arabidopsis thaliana (ATPT2), la 1-désoxyxylulose-5-phosphate synthase (dxs), la réducto-isomérase (dxr), la géranylgéranyl-réductase (GGH), la géranylgéranylpyrophosphate synthase (GGPPS), l'hydroxyphénylpyruvate dioxygénase HPPD, la méthyl-transférase 1 (MT1), la 2-méthylphytylplastoquinol méthyltransférase (TMT2), la tocophérol gamma-méthyl-transférase (GMT), la tocophérol cyclase (slr 1737), et à partir d'une construction antisens pour l'acide homogentisique dioxygénase.
  7. Construction d'acide nucléique comprenant un acide nucléique selon l'une quelconque des revendications 1 à 6.
  8. Protéine comprenant SEQ NO : 2.
  9. Anticorps capable de se lier spécifiquement à une protéine comprenant SEQ ID NO: 2.
  10. Plante transformée comprenant une construction selon la revendication 7.
  11. Plante transformée selon la revendication 10, où ladite plante est choisie parmi la luzerne, Arabidopsis, l'orge, Brassica campestris, Brassica napus, le brocoli, le chou, un agrume, le colza, le coton, l'ail, l'avoine, l'oignon, le lin, une plante ornementale, l'arachide, le poivre, la pomme de terre, le riz, le seigle, le sorgho, la fraise, la canne à sucre, la betterave à sucre, la tomate, le blé, le peuplier, le pin, le sapin, l'eucalyptus, la pomme, la laitue, les lentilles, le raisin, la banane, le thé, le gazon, le tournesol, le soja, le maïs, Phaseolus, le crambe, la moutarde, le ricin, le sésame, le carthame et le palmier à huile.
  12. Plante transformée selon la revendication 10, où ledit promoteur est un promoteur inductible à la lumière ou un promoteur à spécificité tissulaire ou cellulaire, en particulier un promoteur spécifique de la graine ou un promoteur qui fournit l'expression dans un tissu de tubercule, de fruit, de feuille, de graine, d'endosperme, d'ovule, de pollen, d'aleurone, de racine ou de tige.
  13. Plante transformée selon la revendication 12, où ledit promoteur spécifique de la graine est choisi parmi le promoteur de la napine, un promoteur d'inhibiteur de la trypsine du soja, le promoteur de la stéaroyl-ACP désaturase, le promoteur 7S du soja, le promoteur de l'oléosine, le promoteur de la β-conglycinine, un promoteur de la zéine, le promoteur du gène waxy, le promoteur du gène Brittle, le promoteur du gène Shrunken 2, le promoteur pour l'enzyme structurante I, le promoteur pour l'enzyme structurante II, un promoteur d'amidon synthase, un promoteur d'enzymes déstructurantes, un promoteur d'oléosine, un promoteur de glutéline, un promoteur de saccharose synthase, le promoteur de l'Osgt-1, un promoteur de sous-unité de l'ADPglucose pyrosynthase, les promoteurs de l'amidon synthase liée aux granules et d'autres amidon synthases, un promoteur de protéine abondante dans l'embryogenèse, un promoteur de gliadine, un promoteur de gluténine, un promoteur d'hordéine, un promoteur de globuline d'embryon et un promoteur de protéine spécifique de l'aleurone et un promoteur de l'arceline 5.
  14. Procédé de production d'une plante ayant des graines comprenant du tocophérol, le procédé comprenant les étapes consistant à : (A) introduire dans ladite plante une construction telle que définie dans la revendication 7 ; et (B) cultiver ladite plante.
  15. Procédé selon la revendication 14, où ladite plante est choisie parmi la luzerne, Arabidopsis, l'orge, Brassica campestris, Brassica napus, le brocoli, le chou, le colza, un agrume, le coton, l'ail, l'avoine, l'oignon, le lin, une plante ornementale, l'arachide, le poivre, la pomme de terre, le riz, le seigle, le sorgho, la fraise, la canne à sucre, la betterave à sucre, la tomate, le blé, le peuplier, le pin, le sapin, l'eucalyptus, la pomme, la laitue, les lentilles, le raisin, la banane, le thé, le gazon, le tournesol, le soja, le maïs, Phaseolus, le crambe, la moutarde, le ricin, le sésame, le carthame et le palmier à huile.
  16. Graine comprenant une construction telle que définie dans la revendication 7, où ladite graine comprend du tocophérol.
  17. Plante transgénique comprenant la construction telle que définie dans la revendication 7.
EP02769376A 2001-05-09 2002-05-09 Transporteurs de metabolites Expired - Lifetime EP1411761B1 (fr)

Applications Claiming Priority (7)

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US137310 1993-10-15
US28952701P 2001-05-09 2001-05-09
US28951901P 2001-05-09 2001-05-09
US289527P 2001-05-09
US289519P 2001-05-09
US10/137,310 US7238855B2 (en) 2001-05-09 2002-05-03 TyrA genes and uses thereof
PCT/US2002/014445 WO2002090506A2 (fr) 2001-05-09 2002-05-09 Transporteurs de metabolites

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EP1411761A2 (fr) 2004-04-28
WO2002090506A2 (fr) 2002-11-14
EP1411761A4 (fr) 2005-08-17
BR0209536A (pt) 2005-04-26
DE60235168D1 (de) 2010-03-11
BRPI0209536B1 (pt) 2016-05-10
AU2002308633A1 (en) 2002-11-18
WO2002090506A3 (fr) 2004-02-26

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